Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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The material on this page is part of Chapter 10, which is shown in full as a preview on this site.

Chapter 10: Nucleic Acid Platform Technologies

Rando Oliver, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

INTRODUCTION

Amplification of RNA

(Protocol summary only for purposes of this preview site)

Gene expression profiling typically requires microgram quantities of mRNA, which can be difficult to obtain. In such cases, RNA must be amplified in order to have enough material for microarray labeling and hybridization. Currently, the most popular choice for amplifying RNA is to use a commercial kit, such as MessageAmp II (Ambion), a product with which we have had good success. These kits are expensive, however, and thus this protocol provides an alternative RNA amplification procedure adapted from Baugh et al. (2001).


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 Protocol 4: Amplification of RNA

Gene expression profiling typically requires microgram quantities of mRNA, which can be difficult to obtain. In such cases, RNA must be amplified in order to have enough material for microarray labeling and hybridization. Currently, the most popular choice for amplifying RNA is to use a commercial kit, such as MessageAmp II (Ambion), a product with which we have had good success. These kits are expensive, however, and thus this protocol provides an alternative RNA amplification procedure adapted from Baugh et al. (2001).

This protocol generates amplified antisense RNA (aRNA) from limited quantities of total RNA (see Fig. 1). It is designed around maximizing yield and product length while minimizing template-independent side reactions. Template-independent reactions compete with the desired template-dependent reaction, an undesirable situation that becomes more severe as less RNA template is used. Amplification products dominated by template-independent product result in greatly reduced sensitivity and compression of differences in microarray hybridization experiments. Most notably, the oligo(dT) primer used in reverse transcription (RT) yields a high-molecular-weight product in the in vitro transcription (IVT) reaction independent of any cDNA template (Baugh et al. 2001). This reaction occurs under all conditions tested; the protocol is therefore designed to limit the amount of primer used to start with. In addition, high-molecular-weight, template-independent product is generated in the presence of biotinylated NTPs and the absence of any polymer when excessive amounts of T7 RNA polymerase activity are used. Template-dependent product of questionable molecular weight and limited functionality in downstream reactions can also be produced with excessive T7 RNA polymerase activity. Essentially, more yield is not always better. The protocol limits the amount of primer used by employing small cDNA synthesis volumes.

The key consideration in any amplification protocol, as noted in Protocols 2 and 3 for DNA labeling, is preventing representation bias in the amplified material. As with DNA amplification, a valuable initial experiment for investigators who are new to RNA amplification is to compare unamplified RNA and amplified RNA by microarray hybridization. The readout should be designed to reveal which sequences are over- and underrepresented after amplification, and by how much.

At the end of the protocol, quantify the mass yield of amplified material. A single round of amplification typically results in a fivefold to 20-fold mass conversion of starting material. If the first-round aRNA is used as a template for a second round of amplification, 200- to 400-fold amplification is typical.


MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Recipes for reagents specific to this protocol, marked <R>, are provided at the end of the protocol. See Appendix 1 for recipes for commonly used stock solutions, buffers, and reagents, marked <A>. Dilute stock solutions to the appropriate concentrations.

Reagents

  • Carrier
    • Add either 5 g of linear polyacrylamide (LPA) or 20 g of glycogen. If a second round of amplification is to be used (or other downstream reverse transcription reactions), then LPA is recommended over glycogen. LPA will slow the Microcon washes (from 1214 min to 2832 min for Microcon 100s at 500g and room temperature), but it does not inhibit reverse transcriptase.
  • DEPC ddH2O
  • DEPC-treated TE (pH 8.0)
  • DNA ligase, from Escherichia coli
  • DNA polymerase I
  • dNTP (10 mM)
  • DTT (100 mM)
  • (dT)-T7 primer
    • The primer sequence is 5-GCATTAGCGGCCGCGAAATTAATACGACTCACTATAGGGAGA(T)21V-3 (where V stands for A, C, or G).
  • Ethanol (70 and 95)
  • First-strand buffer (5), provided with kit
  • High-yield T7 in vitro transcription kit (e.g., AmpliScribe from Epicentre; similar kits from Ambion, Promega, and others are available)
  • IVT buffer (10), provided with IVT kit
  • NaCl (5 M)
  • NTP mixture (100 mM)
    • The NTP mixture contains 100 mM each of ATP, CTP, GTP, and UTP.
  • Phenol:chloroform
  • Random primers
  • Reverse transcriptase (SuperScript II; Life Technologies)
  • RNase H, from E. coli
  • RNase inhibitor
  • Second-strand synthesis buffer (5) <R> (Lifetech or homemade)
  • T4gp32 (single-strand binding protein; 8 mg/mL)
  • T4 DNA polymerase
  • T7 RNA polymerase, high concentration (80 U/L)
  • Total RNA (100 ng dissolved in water or TE; the concentration does not matter as the RNA will be dried down in Step 1)

Equipment

  • Bio-Gel P-6 Micro-Spin Column (Bio-Rad)
  • Heat block set at 65C and 70C
  • Incubator set at 14C16C
  • Microcon 100 spin column (Millipore)
  • Phase Lock Gel Heavy tubes (0.5 mL) (Eppendorf)
  • Thermal cycler (with a heated lid) or air incubator set at 42C and 70C
  • Tubes (0.6 mL)
  • Rotary evaporator (e.g., SpeedVac)


METHOD
Reverse Transcription

  • 1. Combine 100 ng of the (dT)-T7 primer with 100 ng of total RNA in a 0.6-mL tube. Reduce the volume under vacuum to 5.0 L.
    • Do not allow the RNA to dry out completely.
  • 2. Prepare the RT premix and place it on ice:
  • 3. Denature the RNA/primer mix for 4 min at 70C in a thermal cycler with a heated lid.
  • 4. Snap-cool the mix on ice and keep it on ice.
    • The volume may drop following denaturation.
  • 5. Add 5.0 L of ice-cold RT premix to the RNA/primer tube, and mix by pipetting. The final volume should be 10 L.
    • If there was evaporative loss during denaturation (Step 3), then add dH2O to adjust the volume to 10.0 L. Before committing your RNA, run these initial steps using a control nucleic acid to determine the volume loss.
  • 6. Incubate the RT reaction for 1 h at 42C in either a thermal cycler with a heated lid or an air incubator, but not in a water bath to reduce the chance of contamination.
  • 7. Heat-inactivate the reaction for 15 min at 65C.
  • 8. Chill the tube on ice.

Second-Strand Synthesis

  • 9. Prepare second-strand synthesis (SSS) premix:
    Chill on ice.
  • 10. Add 65 L of ice-cold SSS premix to the RT reaction tube, and mix by pipetting. Incubate for 2 h at 14C16C.
  • 11. Add 2 U (10 U) of T4 DNA polymerase, and mix by flicking and gentle vortexing. Incubate for an additional 15 min at 14C16C.
  • 12. Heat-inactivate the reaction for 10 min at 70C. Go immediately from 15C to 70C without letting the tube sit at room temperature to avoid undesirable enzyme activities.
  • 13. Add 75 L of phenol:chloroform (1:1), and mix by pipetting vigorously. Transfer the mixture to prespun Phase Lock Gel Heavy 0.5-mL tubes, and centrifuge for 5 min at 13,000 rpm.
  • 14. Prepare a Bio-Gel P-6 Micro-Spin Column per the manufacturer's instructions.
  • 15. Transfer the aqueous phase from Step 13 to the prepared P-6 column and centrifuge at 1000g for 4 min, recovering the flowthrough (80 L) in a clean 1.5-mL tube.
    • The flowthrough can be kept in the 1.5-mL tube or transferred to a tube of a different size, depending on the details of the IVT reaction steps. For example, it may be desirable to run the reaction in a thermal cycler; thus, the products can be held at 4C rather than overincubate them.
  • 16. Add the appropriate carrier and 3.5 L of 5 M NaCl for precipitating the DNA, and mix by vortexing. Add 2.5 volumes of 95 ethanol (220 L) and mix well. Precipitate for at least 2 h at 20C.
  • 17. Centrifuge to pellet the DNA at 13,000 rpm for 20 min.
  • 18. Carefully remove the supernatant. Wash the pellet with 500 L of 70 ethanol, and centrifuge for 5 min at 13,000 rpm.
  • 19. Carefully remove the supernatant. Pulse-centrifuge (up to full speed) the tube to collect all residual ethanol at the bottom.
  • 20. Remove the remaining supernatant with a pipette. Allow the pellet to air-dry for 23 min.

In Vitro Transcription

  • 21. Prepare the IVT premix at room temperature to avoid forming a precipitate:
  • 22. Add 40 L of IVT premix to the DNA pellet (from Step 20). Resuspend the pellet in the premix by gently flicking and vortexing the tube. Incubate the reaction for 9 h at 42C.
    • If the starting amount of total DNA was in the microgram range, then IVT yields might improve by using a 60- or 80-L reaction volume.
  • 23. Proceed with the cleanup and additional amplification, or freeze the IVT reaction at 80C.

Cleanup

  • 24. Add 480 L of DEPC-treated TE to the IVT reaction tube.
  • 25. Transfer the 500 L to a Microcon 100, and centrifuge at 500g until the volume is <20 L (1115 min at room temperature without LPA, 2832 min with LPA).
    • Processing a sample through a Microcon spin column is slower with LPA in the sample, although the results are fine. Alternatively, RNeasy columns can be used for cleanup.
  • 26. Add another 500 L of DEPC-treated TE, and centrifuge as before. Repeat this step one more time (three washes total).
    • If you intend to proceed with a second amplification, the final wash should be with dH2O, and with the filtrate should be reduced to a small volume.
  • 27. Measure and, if necessary, adjust the volume for downstream applications.
    • It may be comforting or worthwhile to quantify the yield and analyze the products by electrophoresis. Alternatively, proceed to the second round of amplification.

Second Round of Amplification

  • 28. Add 0.5 g of random primers to the aRNA (from Step 26). Reduce the volume under vacuum to 5.0 L.
  • 29. Denature the RNA for 5 min at 70C in a thermal cycler with a heated lid.
  • 30. Snap-cool the mix on ice. Let the tube rest for 5 min at room temperature.
  • 31. Prepare the RT premix and place it on ice:
  • 32. Add 5 L of room-temperature RT premix to the RNA, and mix by pipetting.
    • The final volume should be 10.0 L. If there was evaporative loss during denaturation (Step 28), then add dH2O to adjust the volume to 10.0 L.
  • 33. Incubate the reaction tube in a thermal cycler with a heated lid, programmed as follows:
    • i. 20 min at 37C.
    • ii. 20 min at 42C.
    • iii. 10 min at 50C.
    • iv. 10 min at 55C.
    • v. 15 min at 65C.
    • vi. Hold at 37C.
  • 34. Add 1 U of RNase H, and mix by vortexing gently. Incubate for 30 min at 37C and then heat for 2 min at 95C.
  • 35. Chill the tube on ice, and then centrifuge it briefly to collect the condensation. Place the tube on ice.
  • 36. Add 1 L of 100 ng/L (dT)-T7 primer while the tube is on ice. Incubate the tube for 10 min at 42C to anneal the primer.
  • 37. Prepare the SSS premix (minus ligase):
  • 38. Snap-cool the sample (from Step 36) on ice.
  • 39. Add 65 L of ice-cold SSS premix to the chilled reaction tube. Incubate for 2 h at 14C16C.
  • 40. Add 10 U of T4 DNA polymerase, and mix by gentle flicking and vortexing. Incubate for an additional 15 min at 14C16C.
  • 41. Heat-inactivate the reaction for 10 min at 70C.
  • 42. Perform phenol:chloroform extraction, Bio-Gel P-6 chromatography, and nucleic acid precipitation as per Steps 1320.
  • 43. Prepare the IVT premix as in Step 21.
  • 44. Add 40 L of IVT premix to the DNA pellet (from Step 42). Resuspend the pellet in the premix by gently flicking and vortexing the tube. Incubate the reaction for 9 h at 42C.
  • 45. Proceed with the cleanup as in Steps 2427, or freeze the IVT reaction at 80C.


DISCUSSION

The output of this protocol will be amplified RNA suitable for direct or indirect labeling for microarray hybridization (Protocols 5 or 6). As with DNA amplification, always quantify the mass yield of amplified material, and include control amplifications without input RNA to ensure that there is no contamination of any of your reagents.

First time users of this protocol will find it helpful to analyze amplified product on denaturing or native agarose gels (see Fig. 1 in Baugh et al. 2001). Identification of high-molecular-weight product in the No Template control likely indicates excess primer, and this can be minimized by decreasing primer concentration in the initial in vitro transcription reactions.

Another useful first-time control is to amplify RNA from an abundant RNA source and to competitively hybridize amplified RNA against the original RNA pool, ideally using a microarray containing probes at the 5 and 3 ends of genes. If the protocol is working well, aRNA and original RNA samples should be highly correlated, and 5/3 ratios should be close to 1. Lower 5/3 ratios indicate poorly processive in vitro transcription, which can be corrected by increasing input RNA mass to the protocol. Alternatively, lower 5/3 ratios may indicate failure to include the single-strand DNA binding protein T4gp32 in the in vitro transcription reaction.


RECIPE

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Second-Strand Synthesis Buffer (5)
REFERENCE
Baugh LR, Hill AA, Brown EL, Hunter CP. . Year: 2001. Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res29: e29. doi: 10.1093/nar/29.5.e29.

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