section 5

5 In vitro transcription

5.1 Synthesis of single-stranded RNA probes

for ISH by the in vitro transcription of double-stranded DNA templates

5.1.1 In vitro transcription

Single-stranded RNA - complementary RNA (cRNA) or riboprobes - can be synthesised in vitro 25,26 using one of the several commercially available phagemids. These contain promoters for the highly specific bacteriophage DNA-dependant RNA polymerases from the Salmonella bacteriophage SP6, and the E. coli. bacteriophages T3 and T7 27. The three polymerases all have similar biochemical properties and consist of a single polypeptide chain of ~ RMM 95 000. Each is highly specific for its own promoter and does not use other bacteriophage, plasmid, bacterial or eukaryotic promoters at a significant rate. They rarely initiate at aberrant sites or nicks in double-stranded DNA templates.

When the template DNA is incubated with the appropriate RNA polymerase, transcription buffer and rNTPs, strand-specific, single-stranded RNA is generated. This can be used for in vitro translation or, if labelled rNTPs are included in the transcription reaction, as probes for filter, solution or in situ hybridisations.

Single-stranded RNA is produced more efficiently than single-stranded DNA and under optimal conditions, 20 moles of RNA are generated per mole of template DNA. The RNA polymerases incorporate > 60% of the radiolabelled rNTP and can function efficiently at low [rNTP] in the range 1 - 20 mM. Large amounts of high specific activity probe can be generated at a reasonable cost.

Single-stranded riboprobes have the additional advantage of hybridising more efficiently to the target mRNA than the comparable double-stranded DNA probe. RNA:RNA duplexes are also more stable than RNA:DNA duplexes and riboprobes tend to produce a stronger hybridisation signal than DNA of the same specific activity.

Most in situ hybridisation applications require relatively high probe concentrations, in the range 50 ng - 1 mg / ml. However, optimal buffer requirements for each enzyme are different 27 and even a small reduction in polymerase efficiency during transcription can lead to a large reduction in the amount of cRNA synthesised 25,26,27,28.

The conditions for in vitro transcription are :

Ľ 40 mM Tris HCl pH 7.9 - 8.0

Ľ 6 mM MgCl2

Ľ 2 mM spermidine HCl

Ľ 10 mM NaCl,

Ľ 10 mM DTT

Ľ 100 mg / ml BSA,

Ľ >250 mM rATP, rCTP, rGTP & rUTP,

Ľ 1 unit / ml of RNasin

Ľ 20 nM linearised plasmid template DNA = 40 mg / ml of a 3 kB plasmid

Ľ 500 - 1000 units / ml of bacteriophage DNA-dependant RNA polymerase

When the concentration of each of the rNTPs is >50 mM, ~ 90% of the cRNA transcripts are full length, and all rNTPs are saturating at 250 mM. rGTP is the initiating nucleotide for SP6 RNA polymerase, which is more tolerant of low [rGTP] than of the other three nucleotides. When used in combination, spermidine at 2 mM and BSA at 100 mg / ml increase the amount of cRNA transcribed by up to 2.5 fold. The mechanism of action is unknown. There is an absolute requirement for both [Mg2+] ions and DTT. SP6 has a pH optimum of pH 7.5 but is relatively insensitive to changes in pH in the range 7.0 - 8.5. The template [DNA] should be > 20 nM which is equivalent to 40 mg / ml of a plasmid 3 kb in length. When the template concentration is > 20 nM and the concentration of each of the rNTPs is > 50 mM the rate of RNA synthesis is linear for over 1 hour and proportional to the amount of enzyme added. Under these conditions, ~ 8 moles of RNA are transcribed from 1 mole of template. If another aliquot of enzyme is added at one hour, this can be increased to 20 moles of RNA per mole of template.

5.1.2 Design of cRNA probes for ISH

Transcription vector

Most transcription vectors are derived from the high copy number pUC (Ampr) plasmids and contain at least one of the three bacteriophage RNA polymerase promoters situated adjacent to a multiple cloning site. Vectors vary in both the choice of promoter and the complexity of the cloning site. Single promoter vectors allow transcription of only one strand but dual promoter vectors allow transcription of both strands of the subcloned insert.

The most commonly used transcription vectors are the pGEM (SP6 and T7 promoters, Promega 29) and pBluescript (T3 and T7 promoters, Stratagene) phagemids. The choice of vector is largely a matter of personal preference - I use pBluescript II (Stratagene) which has dual promoters - T3 and T7 - either side of synthetic polylinker containing 23 unique restriction enzyme sites. These sites generate 5' and 3' overhangs and blunt ends and generally allow the directional cloning of DNA inserts. pBluescript KS II has the polylinker in the reverse orientation to pBluescript SK II. pBluescript carries the regulatory sequences and amino terminus coding region of the E. coli b-galactosidase (lacZ) gene, which allows a-complementation when pBluescript is transformed into a lacZDM15 bacterial host. The polylinker and both flanking promoters are situated within the lacZ coding region. Successful subcloning into the polylinker disrupts lacZ and results in a non-functional peptide. This allows screening by blue (non-recombinant) / white (recombinant) colony selection after induction of lacZ with IPTG and incubation of bacteria on X-gal substrate. pBluescript also contains the bacteriophage M13 origin of replication, and can be used to generate single-stranded DNA complementary to one of the strands of the inserted DNA. I have not had any problems with non-specific hybridisation of pBluescript polylinker sequences 30.

If using both pGEM (SP6 and T7) and pBluescript (T3 and T7) phagemids, it is worth noting that SP6 RNA polymerase is inhibited by high [NaCl] 31. The [NaCl] of pBluescript transcription buffers is 50 mM. The 40 mM TrisCl (pH 8.0), 2 mM spermidine, 6 mM MgCl2 and 10 mM NaCl transcription buffer works well for all 3 RNA polymerases. SP6 RNA polymerase transcribes optimally at 40oC, whereas T3 and T7 both transcribe at 37oC. For this reason, I find pBluescript more convenient to use.

Design of subclone

Which probes work for ISH has to be determined empirically. Several subclones may have to be made before one with a suitable S/N ratio is found. General guides for probe construction are as follows :

Ľ For ISH, subclone an insert in the range 500 - 1 000 bp in length. This is large enough to be sensitive, but small enough to penetrate tissue without prior hydrolysis. Smaller probes will work if the target mRNA is very abundant (eg ~ 70 bp GAP-DH probe) Ľ If the same probe is to be used in RNase protection assays (RPA), the insert DNA should preferably be in the range 100 - 400 bp in length. Full-length cRNA is required for RPA, and it should be the major reaction product after in vitro transcription. However, the low concentration of the radiolabelled rNTP favours premature termination of cRNA transcripts over full-length transcription. This tendency is exacerbated by using large inserts. I have successfully used cRNA of ~650 nucleotides for RPA where the target mRNA was abundant Ľ If the insert is to be used for cold / non-radioactive in vitro transcriptions, the subcloned insert can be several kb long Ľ The enzymes will read through homopolymeric tracts of ~30 bases but they are best removed (if possible) before subcloning eg large poly A+ tracts Ľ If the full cDNA is not required for ISH, subclone a segment from the coding region. cRNA generated from non-coding/repetitive regions tends to produce high backgrounds. Where possible, avoid conserved sequences eg homeodomains. These can produce misleading cross-hybridisation signals. If there is any doubt about the specificity of the cRNA probe, formally test it by RPA using synthetic sense mRNAs Ľ The 5' end of the transcript is fixed by the position of the bacteriophage promoter. The 3' end is determined by the position of the restriction enzyme cleavage site. The length of the plasmid sequences transcribed into any cRNA therefore depends on the distance downstream from the promoter of the inserted foreign DNA. Plasmid sequences in cRNA used for plasmid library screening and for ISH hybridisation may cause high backgrounds. This problem can be eliminated by subcloning the insert as close downstream as possible of the promoter or, if this is not possible, including ÔcoldŐ vector DNA in the prehybridisation and hybridisation buffers Ľ I find T7 to be more consistent and efficient than either of the other two enzymes. I subclone the insert such that the T7 promoter is used to generate the cRNA eg if the construct is intended for generation of an antisense riboprobe, the insert is subcloned with the 3Ő terminus adjacent to the T7 promoter

5.1.3 Choice of radio-label : effect on S/N in ISH

Although RNA polymerases are more tolerant of low [GTP], radiolabelled UTP is the most stable of the four rNTPs 32. For this reason, radiolabelled cRNAs are transcribed using either a-35S, a-33P, a-32P or 5,6H-UTPs

a-35S-UTP-labelled cRNA

Traditionally, the preferred isotope for ISH of radiolabelled cRNA probes to mRNA has been 35S, because of its long half life, high signal with abundant mRNAs and good spatial resolution 33. However, it is the most poorly processed radio-nucleotide when using bacteriophage RNA polymerases 32 and interacts with tissue proteins to produce a unique form of background in ISH 34. This background can be reduced, but not entirely eliminated, by using reducing agents such as DTT. Protocols for ISH with a-35S-UTP-labelled cRNA probes have been designed for tissues with low intrinsic backgrounds, such as brain 35 and embryos 36. 35S gives less acceptable ISH results with protein-rich adult tissues such as pancreas, which have high intrinsic backgrounds. The signal from abundant mRNAs can easily be detected over high, 35S-generated backgrounds. However the small signal from low abundance mRNAs is often obscured.

a-33P-UTP-labelled cRNA

A relatively new b-emitting isotope of phosphorus, 33P, was introduced in early 1992 as an a-UTP. It is efficiently incorporated into cRNA, results in fewer prematurely terminated transcripts and can be used in smaller volume transcription reactions than 35S. For standard X-ray film autoradiography, its properties are intermediate between those of 32P and 35S (below). For liquid film emulsion autoradiography, the resolution and signal generated by 35S and 33P-labelled cRNA are equivalent but the background of 33P is less 37. The S/N ratio of 33P is \ better than that of 35S and allows detection of low abundance mRNAs in protein-rich adult tissues.

Radioisotope	Half life	Maximum energy	Maximum path
		of emission	length (cm)
32P	14 days	1.7	700
33P	25 days	0.25	49
35S	87 days	0.17	24
3H	12 years	0.02	0.5

32P has the greatest sensitivity but poorest resolution, whilst 3H has the greatest resolution but poorest sensitivity

a-32P-UTP-labelled cRNA

The resolution and signal generated by 32P are relatively poor although the background is low. The apparent paradox that the energy of b-emission does not correlate with the autoradiographic signal strength is presumably because much of the high energy b-emission from 32P passes straight through the film emulsion without leaving a latent image.

3H-UTP-labelled cRNA

The resolution of 3H is good enough for subcellular localisation at the electron microscopic level. The background approaches that of the intrinsic background of the liquid film emulsion. b-emission from 3H is surprisingly efficient at producing a latent image, presumably because all the energy of emission is trapped within the layer of film emulsion. The S/N is high. Abundant mRNA targets hybridised with 3H-labelled cRNAs can generate a visible slide autoradiographic signal in as little as 4 - 7 days (BFJ, unpublished data)

Resolution and S/N ratio of different radiolabels

X-ray film	Signal	32P >	35S =	33P >>>	3H
	Resolution	3H =	33P =	35S >>>	32P
	Background	32P >	35S >	33P >	3H
Liquid film emulsion	Signal	33P =	35S >	3H >>>	32P
	Resolution	3H >>>	33P =	35S >>>	32P
	Background	35S >	32P =	33P >	3H

Choice of radiolabels for ISH applications

32P

Ľ Screening for organ or tissue expression using standard X-Ray film

Ľ Slide autoradiography for high abundance mRNAs

Ľ Slide autoradiography for large structures - not single cell resolution

33P

Ľ Screening for organ or tissue expression using standard X-Ray film

Ľ Slide autoradiography for low abundance mRNAs

Ľ Slide autoradiography for resolution of single cells / small groups of cells

35S

Ľ Screening for organ or tissue expression using standard X-Ray film

Ľ Slide autoradiography for medium - high abundance mRNAs in tissues with medium - low

backgrounds

Ľ Slide autoradiography for resolution of single cells / small groups of cells

Ľ Slide autoradiography for high abundance mRNAs

Ľ Slide autoradiography for subcellular resolution

5.1.4 Alkaline hydrolysis

RNA probes of 50 - 200 nucleotides in length have been reported to generate better hybridisation signals than longer probes 38. Many ISH protocols therefore include limited alkaline hydrolysis of labelled cRNA. I and others have found that hydrolysis raises background but not signal. It is also an extra stage during which the RNA probe can become fully degraded. I have successfully used probes of ~ 1 kb without hydrolysis. An additional consideration is that the specificity of the cRNA may be decreased.

5.2 Preparation of template DNA for in vitro transcription

Summary

subclone DNA insert into transcription vector Ż purify plasmid DNA Ż linearise with appropriate restriction enzyme - cut to completion Ż Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ż Remove 3' overhang with T4 DNA polymerase Ż Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ż phenol-chloroform extract x 2 Ż chloroform extract x 1 Ż precipitate with isopropanol / Na acetate Ż wash with 70% ethanol Ż resuspend at 1 mg / ml in sterile MilliQ water - gel check Ż store at -20oC

To produce 'run off' rather than 'run round' transcripts, the vector has to be cut downstream of the promoter being used. The enzyme can cut at multiple sites as long as the promoter is not detached from the insert : the RNA polymerase should only initiate at the promoter. For dual promoter vectors, decide which promoter to use (reading 3' Ž 5' through the insert will generate anti-sense RNA).

Choose a suitable restriction enzyme site downstream of this promoter. It should preferably cut leaving a 5' overhang or a blunt end. Less than 0.1% of the transcripts will initiate on the other strand. If a 3' overhang is generated, the polymerases will transcribe both strands of the insert by :

Ľ initiating on the 3' overhang

Ľ initiating at the promoter and 'turning the corner' at the end of the template

Ľ aberrant (non-promoter) initiation 39.

3' overhangs must first be removed with a 3' Ž 5' exonuclease, such as T4 DNA polymerase, before the template can be used for transcription 40,41.

The template must be cut to completion. Any uncut plasmid will be transcribed to produce 'run round' transcripts which, because of their large size, will consume a significant proportion of the rNTPs. This is a major problem when using radiolabelled rNTPs at low concentrations

Reagents

Appropriate restriction enzymes and buffers Reagents / equipment for ethidium-agarose gel electrophoresis of DNA Phenol Analar grade crystals, BDH, Catalogue number 10188 (500 g) 8-hydroxyquinoline Analar grade crystals, BDH, Catalogue number 10131 (100 g) Sterile MilliQ water Tris base Sigma, UltraPure, Catalogue number T6791 (100g) Sterile 10 mM TrisCl, 1 mM EDTA buffer pH 8.0 Chloroform Analar grade, BDH, Catalogue number 10077 (500 ml) Sodium acetate anhydrous, molecular biology grade, BDH, Catalogue number 44389 (500 g) Isopropanol Analar grade, BDH, Catalogue number 10224 (1 litre / 2.5 litre) 70% ethanol Removal of 3' overhangs T4 DNA polymerase + 10 x buffer Promega, Catalogue number M4212 (100 units) (dATP, dCTP, dGTP, dTTP) dNTP set UltraPure solution dNTPs, Pharmacia, Catalogue number 27-203501 (2 x 25 mmol) Spermidine tetrahydrochloride Sigma, molecular biology grade, catalogue number S2876 (1g)

In advance

1 TE-saturated phenol Melt the phenol crystals overnight in sterile MilliQ water, with 0.1% (w/w) 8-hydroxyquinoline. Wash the phenol twice with an equal volume of 100 mM sterile Tris base. The pH should be ~8.0. Wash with an equal volume of TE buffer and store at 4oC 2 3 M sodium acetate pH 7.0 - make up in sterile MilliQ water, pH with acetic acid and autoclave 3 Prepare plasmid DNA Several methods of plasmid purification can be used but the only absolute requirement is that the plasmid is free of contaminating RNAses. If alkaline lysis mini-preps have been RNaseA-treated, digest the DNA with 300 mg / ml of Proteinase K in 0.5% SDS, 100 mM NaCl, 10 mM Tris Cl pH 7.4 and 1 mM EDTA pH 8.0, at 37oC for ~1 hour. Extract twice with an equal volume of TE-saturated phenol-chloroform (1:1), once with chloroform and then precipitate with one volume of isopropanol and 1/10 volume of 3M sodium acetate pH 7.0. Recover the DNA by centrifugation, wash with 70% ethanol and proceed to restriction enzyme digestion 4 8 mM dNTPs, 2 mM of each, diluted in sterile MilliQ water. Store in small aliquots at -20oC 5 100 mM spermidine : make up with sterile MilliQ water. Store in small aliquots at -20oC

Protocol

1 Digest 10 - 50 mg plasmid DNA with the appropriate restriction enzyme to completion. Confirm by agarose gel electrophoresis of a small aliquot 2 For 5' overhangs Ž 5 For 3' overhangs : Add 1 ml of 8 mM dNTPs per 20 ml of digestion reaction and 1 - 2 units of T4 DNA polymerase per mg of plasmid DNA 3 Incubate for 15 minutes at 12oC 4 Add an equal volume of TE buffer to stop the reaction 5 Dilute the reaction to 100 ml with sterile TE, extract twice with an equal volume of TE-saturated phenol-chloroform (1:1) 6 Extract twice with an equal volume of chloroform 7 Precipitate with one volume of isopropanol and 1/10 volume of 3M sodium acetate pH 7.0 8 Recover the DNA by centrifugation 9 Wash the pellet with 70% ethanol 10 Resuspend at 1 mg / ml in sterile MilliQ water. This is a concentration of 0.5 pmole / ml when the plasmid is 3 kb in length. Confirm the DNA concentration by agarose gel electrophoresis 11 Store at -20oC

Notes

1 Restriction digests resulting in 3' overhangs should be performed in as small a volume as possible. 100 mM spermidine (DNA denaturant) added to a final concentration of 4 mM will accelerate digestion. nb - spermidine will also precipitate the DNA at 4oC 2 At 12oC, the polymerisation and exonuclease activities of T4 DNA polymerase are roughly equivalent. The exonuclease activity is threefold higher than polymerisation at 37oC 3 DNA will partition into the organic phase during phenol-chloroform extraction if the pH of the phenol is < ~7.4

5.3 Synthesis of cRNA probes for in situ hybridisation

Summary - synthesis and characterisation

transcribe cRNA 37oC - T3 & T7 40oC SP6 > 1 hour Ż DNase 1-digest template DNA, 10 mins 37oC Ż dilute to 200 ml with 10 mM DTT Ź Ź Ź Ź Ź Ź Ź Ź Ź Ź Ž Ž Ž Ž Ž Ž Ž Ž Ž Ž Ż Ż Ż 2 ml for PAGE + AR 2 ml for estimate Ż of % age incorporation phenol-chloroform extract x 2 Ż chloroform extract x 1 Ż precipitate with isopropanol / LiCl Ż wash with 70% ethanol Ż resuspend in 100 ml of 5 mM DTT, 1 unit / ml RNasin in sterile MilliQ water Ż Ż Ż 1 ml for incorporation Ż Ż Ż calculate % age incorporation store at -20oC / proceed to ISH

5.3.1 Reagents

Dithiothreitol Sigma, molecular biology grade, Catalogue number D9779 ATP UltraPure 100 mM solution, Pharmacia, Catalogue number 27-2056-01 (25 mmol) CTP UltraPure 100 mM solution, Pharmacia, Catalogue number 27-2066-01 (25 mmol) GTP UltraPure 100 mM solution, Pharmacia, Catalogue number 27-2076-01 (25 mmol) UTP UltraPure 100 mM solution, Pharmacia, Catalogue number 27-2086-01 (25 mmol) a-32P-UTP 3000 Ci / mmol, 10 mCi / ml, BresaTec, aqueous solution a-33P-UTP >1000 Ci / mmol, 20 mCi / ml, Amersham, Catalogue number BF1002 (250 / 500 mCi) a-35S-UTP >1000 Ci / mmol, 10 mCi / ml, Amersham, Catalogue number SJ1303 (500 mCi / 1 mCi) 5,63H-UTP 43 Ci / mmol, 1 mCi / ml, Amersham, 50% ethanol solution, Catalogue number TRK412 (1 mCi) Acetylated nuclease free BSA Promega, Catalogue number R3962 (10 mg) RNasin ribonuclease inhibitor Promega, Catalogue number N2511 (2500 units) TrisCl / Spermidine Sigma Ž pg 27 / pg 37 MgCl2 Sigma, as hexahydrate, Catalogue number M0250 (100 g) NaCl Sigma, molecular biology grade, Catalogue number S3014 (500 g) SP6 RNA polymerase Promega, Catalogue number P1085 (1 000 units) T3 RNA polymerase Promega, Catalogue number P2083 (1 000 units) T7 RNA polymerase Promega, Catalogue number P2057 (1 000 units) RQ1 RNase-free DNaseI Promega, Catalogue number M6101 (1 000 units) Brewer's yeast tRNA Boehringer Mannheim, Catalogue number 109 517, (100 mg) Phenol / hydroxyquinoline BDH Ž pg 37 LiCl Sigma, molecular biology grade, Catalogue number L9650 70% ethanol Sterile MilliQ water

In advance

1 Template DNA solution at 1 mg / ml in sterile MilliQ water 2 250 mM DTT in sterile MilliQ water 3 30 mM rNTP mix (ATP, CTP, and GTP in equimolar ratio) in sterile MilliQ water 4 100 mM UTP in sterile MilliQ water 5 1 mg / ml acetylated nuclease free BSA in sterile MilliQ water 6 5 x transcription buffer 200 mM TrisCl pH 8.0, 30 mM MgCl2, 10 mM spermidine, 50 mM NaCl in sterile MilliQ water. Autoclave and store in small aliquots at -20oC 7 10 mg / ml tRNA Dissolve in sterile MilliQ water, extract twice against acid-phenol:chloroform, isopropanol-LiCl precipitate, wash with 70% ethanol, resuspend in sterile MilliQ water. Store in small aliquots at -20oC 8 Water saturated phenol Melt the phenol crystals overnight in sterile MilliQ water, with 0.1% (w/w) 8-hydroxyquinoline. Wash with an equal volume of sterile MilliQ water. Aliquot and store at -20oC 9 8 M LiCl in sterile MilliQ water. Autoclave before use

Notes

1 DEPC-contamination of the MilliQ water will inhibit the RNA polymerases. Use autoclaved glassware, make up solutions with autoclaved MilliQ water and re-autoclave where possible 2 DNA will partition into the organic phase and RNA into the aqueous phase during phenol-chloroform extraction with acid-phenol 3 LiCl will preferentially precipitate RNA rather than DNA, from solution 4 a-33P-UTP, a-35S-UTP and 5,63H-UTP from Amersham have performed consistently

5.3.2 Protocols

1 At * 22oC * add :

a-32P-UTP-labelled cRNA

Ľ 0.4 ml 1 mg/ ml template DNA = Final concentration of ~40 mg / ml Ľ 0.4 ml 250 mM DTT = ~10 mM Ľ 0.25 ml 30 mM rNTP mix = 700 mM Ľ 1 ml 100 mM rUTP = 9.25 mM Ľ 1 ml 1 mg / ml BSA = ~100 mg / ml Ľ 0.25 ml RNasin = ~1 unit / ml Ľ 2 ml 5x transcription buffer = 1 x Ľ 5 ml a-32P-UTP : 3000 Ci / mmol, 10 mCi / ml 50 mCi / reaction = 1.67 mM Total [UTP] = ~11 mM Ľ 0.5 ml RNA polymerase = 5 - 10 units S 10.8 ml

a-33P-UTP-labelled cRNA

Ľ 0.4 ml 1 mg/ ml template DNA = Final concentration of ~40 mg / ml Ľ 0.4 ml 250 mM DTT = ~10 mM Ľ 0.25 ml 30 mM rNTP mix = 680 mM Ľ 0.25 ml 100 mM rUTP = 2.3 mM Ľ 1 ml 1 mg / ml BSA = ~100 mg / ml Ľ 0.25 ml RNasin = ~1 unit / ml Ľ 2 ml 5x transcription buffer = 1 x Ľ 1 ml a-33P-UTP : 1000 Ci / mmol, 20 mCi / ml 20 mCi / reaction = 2 mM Total [UTP] = 4.3 mM Ľ 4.95 ml sterile MilliQ water = Ľ 0.5 ml RNA polymerase = 5 - 10 units S 11 ml

a-35S-UTP-labelled cRNA

Ľ 1 ml 1 mg/ ml template DNA = final concentration of 40 mg / ml Ľ 1 ml 250 mM DTT = 10 mM Ľ 0.75 ml 30 mM rNTP mix = 900 mM Ľ 2.5 ml 1 mg / ml BSA = 100 mg / ml Ľ 0.75 ml RNasin = 1.2 unit / ml Ľ 5 ml 5x transcription buffer = 1 x Ľ 2 ml 100 mM rUTP = 8 mM Ľ 10 ml a-35S-UTP : 1000 Ci / mmol, 10 mCi / ml 100 mCi / reaction = 4 mM Total [UTP] = 12 mM Ľ 1 ml sterile MilliQ water = Ľ 1 ml RNA polymerase = 10 - 20 units S 25 ml

5,6 3H-UTP-labelled cRNA

Ľ Dry down 25 ml 5,63H-UTP : 43 Ci / mmol, 1 mCi / ml 25 mCi / reaction = 28.5 mM Resuspend in : 13 ml sterile MilliQ water Ľ 1 ml 1 mg/ ml template DNA = final concentration of 40 mg / ml Ľ 1 ml 250 mM DTT = 10 mM Ľ 0.75 ml 30 mM rNTP mix = 900 mM Ľ 2.5 ml 1 mg / ml BSA = 100 mg / ml Ľ 0.75 ml RNasin = 1.2 unit / ml Ľ 5 ml 5x transcription buffer = 1 x Ľ 1 ml RNA polymerase = 10 - 20 units S 25 ml 2 Incubate at 37oC (for T3 and T7) or 40oC (for SP6) for at least 1 hour, or overnight 3 Add 1 ml of DNase I and incubate at 37oC for 10 minutes 4 Add 10 mM DTT in sterile water to a final volume of 200 ml - use 2 ml for polyacrylamide gel electrophoresis - use 2 ml for estimation of radio-nucleotide incorporation 5 Extract twice with an equal volume of acid-phenol:chloroform 6 Extract once with an equal volume of chloroform 7 Add 10 ml of carrier tRNA and precipitate with an equal volume of isopropanol and 1/10 volume of 8 M LiCl 8 Recover the cRNA by centrifugation at 12 000g for 15 minutes 9 Wash the pellet with 70% ethanol 10 Resuspend in 100 ml of 5 mM DTT, 1 unit / ml RNasin in sterile MilliQ water 11 Remove a further 1 ml aliquot for calculation of cRNA yield (5.4.2 pg 44) 12 Either proceed to ISH, or store at -70oC

Notes

1 Ratio of [labelled-UTP] : [cold UTP]. The conditions chosen for 32P, 33P and 35S are a good compromise between full length transcription and high specific activity. From RNase protection assay data, hotter probes radiolyse faster and are not any more sensitive. The tritiated [UTP] is high enough not to need additional cold UTP 2 Relationship between specific activity, mCi and pmoles

Specific activity		Figures in picomoles
mCi / mmole		mCi
Ci / mmol	1	10	50	100
10		100	1000	5000	10000
100		10	100	500	1000
500		2	20	100	200
1000	1	10	50	100
3000	0.33	3.3	16.7	33

3 Concentration of a-xN-UTP in a reaction in relation to its specific activity and its radioactive concentration

Radioactive	Concn of	a-xN-UTP	in the	reaxn mixture	mM
Concentration		Specific activity
mCi / ml	1000	2000	3000	4000	5000
0.1		0.1	0.05	0.03	0.025	0.02
0.5		0.5	0.25	0.17	0.12	0.10
1.0		1.0	0.5	0.33	0.25	0.20
5.0		5.0	2.5	1.67	1.25	1.00

5.4 Characterisation and purification of cRNA probes

5.4.1 Denaturing polyacrylamide gel electrophoresis

A small aliquot of the transcription reaction is analysed by denaturing polyacrylamide gel electrophoresis. The gel is dried and then autoradiographed 42,43,44

Reagents

Acrylamide BioRad, Catalogue number 161 0107 (1 kg) Bisacrylamide BioRad, Catalogue number 161 0201 (50g) Urea BioRad, Catalogue number 161 0731 (1 kg) Tris base, EDTA Sigma Ž pg 37, pg 27 Boric acid Sigma, molecular biology grade, Catalogue number B6768 (500 g) Ammonium persulphate Sigma, molecular biology grade, Catalogue number A9164 (25 g) TEMED Sigma, molecular biology grade, Catalogue number T7024 (25 ml) Formamide Merck, proanalysi, Catalogue number 1.09684 (1 litre) Bromophenol blue BioRad, Catalogue number 161 0404 (10 g) Sigmacote Sigma, Catalogue number SL-2 (100 ml)

Equipment

15 cm x 15 cm glass plates, gel combs and spacers, vertical electrophoresis tank 20 ml syringe and 21 gauge needles Duck-billed tips Stratagene, Flat 0.17 mm, 0.1 - 10 ml Catalogue number #410005 (or equivalent) Whatmann paper or equivalent Glad Wrap Gel dryer X-ray film / autoradiographic cassettes

In advance

1 Clean the glass gel plates Ž KOH in methanol (5 g / 100 ml) if necessary Ž distilled water Ž 70% ethanol Ž 100% methanol Ž Sigmacote. Clean the gel comb and spacers 2 40% acrylamide solution 38% (w/v) acrylamide, 2% (w/v) bis-acrylamide dissolved in sterile MilliQ water. Filter through a 0.2 mm filter and store in dark glass at 4oC 3 10 x TBE : 890 mM Tris-borate, 20 mM EDTA, pH ~ 8.1 - 8.3. Add 108 g of Tris base, 55 g of boric acid and 40 ml of 500 mM EDTA pH 8.0 to 800 ml of sterile MilliQ water. Make up to 1 litre and auto-clave 4 Fresh 10% (w/v) ammonium persulphate solution in sterile MilliQ water 5 Formamide loading buffer 80% deionised Merck formamide, 1 x TBE and 1/10 volume of saturated aqueous bromophenol blue solution

Protocol

1 Dissolve urea to a final concentration of 7M in 6 - 8% acrylamide and 1 x TBE. Make up ~ 25 ml for 15 cm x 15 cm x 1 mm gels. Mix on a rotating wheel until all the urea has dissolved 2 Add 100 ml of 10% (w/v) ammonium persulphate per 10 ml of acrylamide - urea solution and mix by inversion 3 Add 10 ml of TEMED per 10 ml of acrylamide - urea solution and mix by inversion 4 Cast the gel using a syringe and insert the gel comb, taking care not to introduce bubbles around the teeth. Leave to polymerise for 45 - 60 minutes at 22oC. A sharp schlieren line should be visible around the teeth of the comb if polymerisation has occurred properly. The gel should be used immediately for RNA analysis 5 Remove the comb carefully and rinse out the urea and fragments of un-polymerised acrylamide from the wells with 1 x TBE and a syringe with 21 gauge needle 6 Clip the plates into a vertical electrophoresis apparatus (notched back-plate against the top cathode chamber), fill the tank with 1 x TBE buffer and pre-electrophorese for 10 - 15 minutes at ~450 V 7 Add 4 ml of formamide loading buffer to the 2 ml cRNA samples and vortex. Heat denature for 2 - 3 minutes at 80oC and then chill on ice 8 With the power off, rinse out the wells again with a syringe until no urea can be seen floating into the cathode chamber buffer. Load the samples carefully into the bottom of the well using thin duck-billed tips 9 Electrophorese at ~450 - 500V until the BPB dye front has migrated to the bottom of the glass plates Ž ~ 1 hour In a 10% polyacrylamide gel, a 77 bp fragment is ~ 1/2 of the way down the gel when the BPB dye front just reaches the bottom 10 Prise the gel plates apart and lift the gel onto Whatmann paper or equivalent. Cover the gel in glad wrap and then dry on a commercial gel dryer at 80oC for up to 2 hours 11 Remove the dried gel and peel off the glad wrap. Autoradiograph overnight at 22oC

Notes

1 Acrylamide is a potent, skin-absorbed neurotoxin. 2 Acrylamide and bis-acrylamide are slowly deaminated to acrylic acid ; the reaction is catalysed by light and alkali. Check the pH of the solution (neutral) and keep it dark and cool. Re-make solutions every few months. Use sequencing grade reagents - cheaper grades of acrylamide often contain contaminants 3 Discard 10 x TBE buffer when a white precipitate forms 4 Deionise the formamide with a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the formamide, after deionisation, has a pH is >7.5, discard it 5 Acrylamide-urea gels cast the day before and kept in the fridge tend to produce smeary RNA bands - ? urea / acrylamide decomposition ? - make the gel on the day it is to be used 6 Gels containing cRNAs labelled with 35S generally have to be dried for autoradiography - a wet gel absorbs too much of the signal. Gels for 32P and 33P-labelled cRNAs do not have to be dried although the image is sharper

5.4.2 Estimation of yield

The maximum theoretical yield for each transcription reaction is calculated by assuming 100% incorporation of the radio-labelled UTP and assuming that the average RMM of each rNTP is 330. For the transcription reactions detailed in 5.3.2 pg 41 =

a-32P-UTP-labelled cRNA

Final [UTP] of 9.25 mM cold + 1.67 mM hot = 10.92 mM This = 10.92 pmol UTP / ml = 3.6 ng of UTP (330 pg of rNTP = 1 pmole) \ 100% incorporation of UTP generates 4 x 3.6 ng = 14.4 ng cRNA / ml \ a 10.8 ml reaction generates ~155 ng of cRNA

a-33P-UTP-labelled cRNA

Final [UTP] of 2.3 mM cold + 2 mM hot = 4.3 mM This = 4.3 pmol UTP / ml = 1.4 ng of UTP (330 pg of rNTP = 1 pmole) \ 100% incorporation of UTP generates 4 x 1.4 ng = 5.6 ng cRNA / ml \ an 11 ml reaction generates ~62 ng of cRNA

a-35S-UTP-labelled cRNA

Final [UTP] of 8 mM cold + 4 mM hot = 12 mM This = 12 pmol UTP / ml = 3.9 ng of UTP (330 pg of rNTP = 1 pmole) \ 100% incorporation of UTP generates 4 x 3.9 ng = 15.6 ng cRNA / ml \ a 25 ml reaction generates ~390 ng of cRNA

5,6 3H-UTP-labelled cRNA

Final [UTP] of 28.5 mM This = 28.5 pmol UTP / ml = 9.4 ng of UTP (330 pg of rNTP = 1 pmole) \ 100% incorporation of UTP generates 4 x 9.4 ng = 37.6 ng cRNA / ml \ a 25 ml reaction generates ~940 ng of cRNA

The actual yield is then calculated by measuring the percentage incorporation of the radiolabelled UTP.

The 2 / 200 ml aliquot removed after transcription but before precipitation is dotted onto a Whatmann glass micro fibre filter, called filter A, and allowed to air dry. The 1 / 100 ml aliquot removed from the precipitated, resuspended cRNA is similarly dotted onto a glass filter, called filter B. Both filters are counted at a fixed distance using a hand-held b-counter. Sample A represents all radiolabelled UTP in the reaction. Sample B is largely incorporated radiolabelled UTP since nucleoside triphosphates are poorly precipitated with alcohols 45.

Percentage incorporation = counts filter B x maximum theoretical yield

counts filter A

Although a relatively crude method of assessing cRNA yield, it has been adequate for all my ISH work to date.

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This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/21/95.

25 D. A. Melton, P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn and M. R. Green (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter Nucleic Acids Res 12:7035-7056i 26 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415 27 M. J. Chamberlin and T. Ryan (1982) Bacteriophage DNA-dependent RNA polymerases Boyer, P.D. ed The Enzymes XV:87 - 108 Academic Press, New York 28 An increase in cRNA yield using a high-capacity HEPES-KOH buffer : V. V. Gurevich, I. D. Pokrovskaya, T. A. Obukhova and S. A. Zozulya (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases Anal Biochem 195:207-213 29 D. E. Titus (1991) Promega Protocols and Applications Guide, Second Edition 30 H. Wittkiewicz, M. E. Bolander and D. R. Edwards (1993) Improved design of riboprobes from pBluescript and related vectors for in-situ hybridisation BioTechniques 14:458 31 E. T. Butler and M. J. Chamberlin (1982) Bacteriophage SP6-specific RNA polymerase. I. Isolation and characterization of the enzyme J Biol Chem 257:5772-5778 32 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415 33 G. Niedobitek and H. Herbst (1991) Applications of in situ hybridisation International Review of Experimental Pathology. Ed Richter, G.W. Solez, K. 32:1 - 56. Harcourt Brace Jovanovitch 34 C. E. Bandtlow, R. Heumann, M. E. Scwab and H. Thoenen (1987) Cellular localisation of nerve growth factor synthesis by in situ hybridisation EMBO J 6:891 - 899 35 D. M. Simmons, J. L. Arriza and L. W. Swanson (1989) A complete protocol for in situ hybridisation of messenger RNAs in brain and other tissues with radio-labelled single-stranded RNA probes J Histotechn 12:169 - 181 36 P. V. Senior, D. R. Critchley, F. Beck, R. A. Walker and J. M. Varley (1988) The localization of laminin mRNA and protein in the postimplantation embryo and placenta of the mouse: an in situ hybridization and immunocytochemical study Development 104:431-446 37 B. E. Faulkner-Jones (1993) 33P : advantages for in situ hybridisation Amersham Life Science 11:5 - 6 38 K. H. Cox, D. V. DeLeon, L. M. Angerer and R. C. Angerer (1984) Detection of mRNAs in sea urchin embryos by in situ hyrbdisation using asymetric RNA probes Dev Biol 101:425 39 E. T. Schenborn and R. C. J. Mierendorf (1985) A novel transcription property of SP6 and T7 RNA polymerases: dependence on template structure Nucleic Acids Res 13:6223-6236 40 J. Sambrook, E. F. Fritsch and T. Maniatis (1989) Molecular Cloning, A Laboratory Manual 41 D. E. Titus (1991) Promega Protocols and Applications Guide, Second Edition 42 J. Sambrook, E. F. Fritsch and T. Maniatis (1989) Molecular Cloning, A Laboratory Manual 43 J. H. Pringle, P. V. Senior and A. Warford (1989) In situ hybridisation to cellular mRNA using single-stranded cRNA probes labelled with 35S In situ hybridisation course. Course manual. Revised 1990 44 L. G. Davis, M. D. Dibner and J. F. Battey (1986) Basic Methods in Molecular Biology 45 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415