Reverse Ligation Mediated RT - 

This procedure was first described by Bertrand et al ( Proceedings of the National Academy of Science USA (1993) Vol. 90 pp. 3496-3500, and Nucleic Acids Research (1994) Vol. 22 pp. 293-300) to demonstrate that ribozymes could be enzymatically active in vivo . We adapted the method to show that certain oligodeoxynucleotides could direct the activity of endogenous ribonuclease H to cleave target mRNA in intact human leukaemia cells following streptolysin O - mediated delivery ( method ) of the antisense effectors ( reference 1 , reference 2 ). The theory of reverse ligation mediated RT - PCR (RL-PCR) is described elsewhere in these pages.

RL-PCR can be broken down into a number of simple steps. Synthesis by in vitro transcription and purification of an RNA linker species. Extraction of total RNA from cells into which oligodeoxynucleotide has been delivered by, for example, streptolysin O permeabilization. Ligation of the RNA linker to all available 5' monophosphates in the purified total cellular RNA sample. Reverse Transcription of the RNA using a gene - specific primer. Amplification of specific fragments by PCR using linker - specific and gene - specific primers. Sub - amplification of the first PCR product using linker specific and nested, labelled, gene specific primers. Alternatively the first PCR may be sequenced using the nested, labelled, gene - specific primer.

Reagents/Solutions

• 5µM Template for linker RNA transcript (duplex of RLPCR oligonucleotides 1and 2) in 30mM NaCl, 2mM Tris - HCl, 0.2mM EDTA pH 8.0.
• 5x Transcription Buffer.
  • 0.2M Tris - HCl pH 7.5
  • 50mM NaCl
  • 10mM spermidine
  • 30mM MgCl ₂
• 0.1M dithiothreitol
• 1M MgCl ₂
• Bacteriophage T7 RNA polymerase (200U/µl)
• RNase Inhibitor (~40U/µl)
• 100mM NTPs
• 20% Acrylamide - bis (19 : 1), 1x TBE buffer.
• 4M guanidine thiocyanate, 5mM sodium citrate pH7, 0.5% sarkosyl
  • To 50g Guanidine thiocyanate add
  • 58.6ml H ₂ O
  • 3.52ml 0.75M sodium citrate pH 7.0
  • 5.28ml 10% sarkosyl
• 2M sodium acetate pH4
• 2-mercaptoethanol
• water saturated phenol pH 4.0 - 4.3
• Chloroform:isoamyl alcohol (49 : 1 by volume)
• Isopropanol
• T4 RNA ligase (6U/µl) + 10 x reaction buffer ( Boehringer ).
• RNase Inhibitor (RNase Block I 40U/µl, Stratagene ).
• DNase I (20 - 30U/µl) ( Boehringer ).
• Linear polyacrylamide (2.5µg/µl, Gaillard & Strauss, Nucleic Acids Research 1990 Vol. 18 pp. 378).
• 10mM EDTA pH 8.0.
• Pfu polymerase (native) + 10 x reaction buffer 1 ( Stratagene ).
• Superscript RTase (Gibco).
• Gene - Specific reverse transcription (RT) primer.
• dNTPs 10mM each.
• RNase A 1ng/µl (boiled)
• RNA Linker specific primer (RLPCR oligonucleotide 3).
• Nested gene specific primer (GSP).
• Labelled, nested, second gene specific primer (DigP).
• Sequencing gel loading buffer (95% formamide, 10mM NaOH, 0.05% bromophenol blue, 0.05% xylene cyanol)
• Sequencing gel mix (6 - 8% polyacrylamide : bis (19 : 1), 7M urea, 1x TBE buffer)
• Sequencing Grade Taq + 5x reaction buffer ( Promega ).
• d/dd NTP mixes (20µM dATP, dCTP, dTTP, 7-deaza dGTP: A mix 350µM ddATP, C mix 200µM ddCTP, G mix 30µM ddGTP, T mix 600µM ddTTP).
• Ethanol, ice cold.
• Light mineral oil.

Transcription and purification of RNA linker

1. Hybridize RLPCR1 (5' ... TTT CAG CGA GGG TCA GCC TAT GCC CTA TAG TGA GTC GTA TTA ...3') and RLPCR2 (5'... TAA TAC GAC TCA CTA TAG ...3').
   1. Add 100µl 100µM RLPCR1 to 100µl 100µM RLPCR2 and 50µl 1x TEN (150mM NaCl, 10mM Tris - HCl, 1mM EDTA pH 8.0)
   2. Heat to 70°C for 5 minutes, allow to cool to room temperature slowly.
   3. Analyse a small aliquot by non - denaturing polyacrylamide gel electrophoresis to ensure that hybrid has been formed.
   4. Dilute hybrid to 5µM by addition of 1750µl 0.2x TEN
2. Transcription reaction. (These are the conditions of Milligan et al Nucleic Acids Research 1987 Vol. 15 pp. 8783-8798). Place the following in an eppendorf in the following order.
   1. 153µl H ₂ O
   2. 50µl 0.1M dithiothreitol
   3. 7µl 1M MgCl ₂
   4. 100µl 5x transcription buffer
   5. 20µl 100µM ATP
   6. 20µl 100µM CTP
   7. 20µl 100µM GTP
   8. 20µl 100µM UTP
   9. 20µl 5µM RLPCR1/RLPCR2 hybrid
   10. 20µl RNase inhibitor (~40U/µl)
   11. 50µl T7 RNA polymerase (200U/µl)
3. Incubate transcription reaction for 6 hours at 37°C.
4. Fractionate reaction template and products by electrophoresis through a 20% non - denaturing polyacrylamide gel.
5. Identify the transcript by UV - shaddowing over a thin layer chromotography plate. Cut out the bands.
6. Mash the gel slices which contain the linker transcript.
7. Extract the linker RNA from the gel by constant agitation in ~ 2 volumes of lysis buffer (20ml 4M GuCSN buffer + 2 ml 2M sodium acetate pH 4.0 + 200µl 2-mercaptoethanol) overnight. Centrifuge (10 - 15,000g, 10 minutes) to pellet the mashed gel. Collect the supernatent.
8. Re - extract the mashed gel slices with the same volume of lysis buffer as above, but for 2 hours.
9. Pool the collected supernatent and precipitate the RNA by addition of 1 volume of isopropanol followed by incubation at -20°C overnight.
10. Pellet the precipitated RNA linker by centrifugation at 10 - 15,000g for 30 minutes.
11. Resuspend the RNA in 550µl lysis buffer and treat as if purifiying total RNA from cells.
12. Resuspend the resultant RNA pellet in 100µl H ₂ O and divide into 10x 10µl aliquots.
13. Store at -20°C until required.

Ligation of linker RNA to total cellular RNA

1. To each reaction tube add 1µg of total cellular RNA in 4µl ddH2O. Store on ice.
2. Make mastermix containing the following per reaction:
   • 1µl 10x ligase buffer,
   • 1-2µl gel purified 25-mer RNA (~500ng)
   • 1µl RNase inhibitor
   • 0.5µl T4 RNA ligase (6U/µl)
   • 0.5µl DNase I (~25U/µl)
   • adjust volume to 6µl/reaction with H ₂ O (1-2µl)
3. Add 6µl of mastermix to each reaction tube.
4. Incubate overnight at 17°C.
5. Incubate at 37°C for 30 minutes (ensures ~ complete DNase I digestion).
6. Stop reaction by addition of 5µl 10mM EDTA pH 8.0 and 200µl of modified lysis buffer (lysis buffer:phenol pH4.3 1:1 by volume), which contains 5µg linear polyacrylamide (co - precipitant) per 200µl. Vortex briefly.
7. Induce phase separation by addition of 25µl chloroform.
8. Recover the upper aqueus phase to a thermal cycler tube and precipitate nucleic acids with 1 volume (ca. 130µl) propan-2-ol. Store at -20°C overnight

Reverse Transcription

1. Spin down precipitates at 10 - 15,000g for 30 minutes. Remove and discard supernatant. Brief second spin, pulse to full speed then release, discard the 2-4µl of supernatant so collected.
2. Resuspend the precipitates in 4µl H ₂ O. Store on ice until ready to proceed.
3. Make RT mastermix 1 with the following components and with this volume per reaction
   1. 0.9µl H ₂ O
   2. 0.7µl Pfu polymerase buffer 1
   3. 0.7µl 0.1M dithiothreitol
   4. 0.2µl 10µM gene - specific RT primer
   5. 0.5µl RNase Block I
4. Heat RNA solutions to 95°C for 5 minutes then place immediately at 37°C. Incubate for 2-3 minutes.
5. Add 3µl of RT mastermix 1 per reaction.
6. Incubate at 37°C for 45 minutes.
7. Make RT mastermix 2 with thefollowing volumes per reaction.
   1. 1.4µl H ₂ O
   2. 0.3µl Pfu polymerase buffer 1
   3. 0.3µl 0.1M dithiothreitol
   4. 0.5µl 10mM dNTPs (each)
   5. 0.5µl Superscript RTase
8. Add 3µl RT mastermix 2 per reaction.
9. Incubate at 37°C for 45 minutes.
10. Incubate at 95°C for 10 minutes.
11. Add 1µl RNase A.
12. Incubate at 37°C for 20 minutes.
13. Store on ice until ready for PCR1.

First PCR

1. Make PCR1 mastermix with the following volumes per reaction.
   1. 4.8µl H ₂ O
   2. 1µl Pfu polymerase buffer 1
   3. 2µl 10µM RLPCR3 (linker specific primer, 5'... GGG CAT AGG CTG ACC CTC GCT GAA A ...3')
   4. 2µl 10µM GSP
   5. 0.2µl Pfu polymerase (~0.5U)
2. Add 10µl PCR1 mastermix to each RT reaction.
3. Overlay with 50µl light mineral oil.
4. Thermal cycle: ~20 cycles.

Second PCR

1. Make PCR2 mastermix with the following volumes per reaction.
   1. 9.5µl H ₂ O
   2. 1.5µl Pfu polymerase buffer 1
   3. 1.5µl 10µM RLPCR3
   4. 2µl 10µM labelled primer
   5. 0.3µl 10mM dNTPs
   6. 0.2µl Pfu polymerase
2. Place 15µl PCR2 mastermix into fresh thermal cycler tubes.
3. Overlay with 50µl light mineral oil.
4. Add 5µl PCR1 reaction.
5. Thermal cycle: ~ 5 to 10 cycles.
6. Remove 2µl to 8µl sequencing gel loading Buffer.
7. Heat to 95°C for 10 minutes.
8. Quench on ice, store on ice until analysed.
9. Fractionate through 6 - 8% polyacrylamide sequencing gel (gel temperature 50°C).
10. Standard detection procedure for your label type. Digoxigenin or fluorescein labelled primers will require the nucleic acids in the gel to be electroblotted or diffusion blotted onto nylon membrane prior to chromogenic detection ( method ).

RL-PCR Sequencing

Please note this protocol is an adaption of the ( Promega ) f mole sequencing procedure.

1. Remove 2µl of PCR1 to 18µl H ₂ O.
2. Place 2µl of the d/dd mixes into each of four tubes (ie an 'A' tube, a 'C' tube etc.)
3. Sequencing mix:
   1. 8µl H ₂ O
   2. 5µl 5x sequencing reaction buffer
   3. 2µl 1µM labelled primer
   4. 1µl of the diluted PCR1reaction
   5. 5U Promega sequencing grade Taq polymerase
4. Add 4µl of sequencing mix to d/ddNTP tubes.
5. Overlay with 25µl light mineral oil.
6. Thermal cycle: ~ 30 cycles.
7. Add 4µl sequencing gel load buffer, vortex and centrifuge to mix. Incubateat 90°C for > 5 minutes.
8. Load hot samples onto standard sequencing gel which has been pre - heated to 50°C.
9. Fractionate by electrophoresis.
10. Standard detection procedure for your label type.