Reverse Transcription

Abstract

Among the methods currently available for gene expression analysis, RT-PCR-based assays are the most suitable approaches for assessing mRNA expression levels in formalin-fixed and paraffin-embedded (FFPE) tissues because they allow the detection of target genes even when using relatively poor or degraded RNA. The reliability of gene expression results, however, can be affected by several technical sources of variability, most of which are referred to the reverse transcription step. This problem is further exacerbated when RNA from FFPE material is used, especially for the presence of inhibitory components and for the often unpredictable RNA degradation levels. In this chapter two alternative reverse transcription methods are described. They have been adapted to RNA from archival material and are useful when optimization of the assay is required because they are almost always based on home-made reagents. The protocols included in this chapter differ mainly for the nature of the reverse transcriptase and the priming strategy used. The choice of the most appropriate method depends especially on the amount of starting RNA and on the expected expression levels of the gene under investigation.

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1 Introduction and Purpose

Formalin fixation and paraffin embedding has been the method of choice for the processing and long-term storage of diagnostic tissue samples for a century. A number of studies has shown that such archival material is in principle amenable to the analysis of mRNA and several protocols have been established for the retrieval of mRNA from routinely processed paraffin-embedded tissues [1–3]. Several methods are available to analyze mRNA expression in tissues, including Northern hybridization, subtractive hybridization, RNase protection assay, and cDNA microarrays. However, these methods are significantly limited by the requirement of fresh, unfixed tissues to permit isolation of abundant and high-quality mRNA. Conversely, reverse transcription-polymerase chain reaction (RT-PCR) offers a number of advantages because it allows the use of relatively small amounts of molecules that may be fragmented or degraded, such as those obtained from embedded tissues. RT can be primed with oligo-dT, but this approach is not suitable in formalin-fixed and paraffin-embedded (FFPE)-derived material because of the degradation and loss of polyA tails during fixation. For this reason, random oligonucleotides or specific antisense primers are recommended when analyzing these tissues. Comple­mentary DNA synthesis from RNA is a critical step in gene expression analysis and it is a major source of variability for all RT-PCR assays when these latter need to be performed in a quantitative manner (RT-qPCR). In fact, reverse transcription yield can vary up to 100-fold depending on reverse transcriptase [4], priming strategy [5], and quantity of RNA used [6], and the variation can also be gene dependent [4, 5]. The technical variability is further exacerbated when using RNA from FFPE samples due to intrinsic tissue heterogeneity, to the presence of inhibitory copurified components [7], and to variable degradation of extracted RNA. Standardization of the assays is necessary to allow the analysis of gene expression at the RNA level in standard diagnostic specimens in a clinical setting. The RT-qPCR assay can be performed combining both RT and PCR enzymes in the same tube (single-step RT-PCR system), using an enzyme endowed with both RT and polymerase activity (as the Tth polymerase: one enzyme/one tube system) or separating RT and amplification steps (two-step RT-PCR). Furthermore, commercial RT kits or home-made reagents can be used. We think that higher sensitivity and potential for optimization make the use of the two-enzyme protocol the best choice especially when using home-made reagents [8].

Herein we propose two protocols which differ for the type of reverse transcriptase and for the priming strategy used. The choice of the protocol depends mainly on RNA availability and on the expected expression levels of the target genes.

2 Reverse Transcription with MMLV-RT and Random Hexamers

This protocol combines the use of MMLV-RT (Moloney Murine Leukemia Virus Reverse Transcriptase) with “random priming” strategy. Random hexamers prime RT at multiple points along the transcript; this method is nonspecific but yields the most cDNA and is the most useful for transcripts with significant secondary structures. Furthermore, this strategy generates the least bias in the resulting cDNA, because the same reverse transcription reaction can be used for the detection of several genes in the same sample [9]. It is not the method of choice if the mRNA target is present at low levels, as less abundant targets appear to be reverse transcribed less efficiently than more abundant ones: this is probably due to the lower specific efficiency or to aspecific priming of ribosomal RNA [9]. However, when the starting RNA is low, a specific reverse transcriptase with reduced RNase H activity¹ (e.g., MMLV-RT RNase H minus, Superscript^TM) can be used. Moreover, recent results obtained by one of the IMPACTS groups showed a clear improvement of RT yield when using high concentration of random primers (3.35 nmol/reaction) in the reaction with MMLV enzyme [10].

The RT reaction step involving MMLV and random hexamers has been optimized specifically for quantitative studies in FFPE samples. This method improves the detection of scarcely expressed genes in paraffin-embedded tissues and should allow comparable results between fresh and fixed samples [11]. Such protocol is described as follows.

2.1 Reagents

Reagents from specific companies are reported here, but reagents of equal quality purchased from other companies may be used.

All reagents should be RNase free or DEPC treated. Refer to Chapter “RNA extraction from FFPE Tissues” for precautions against RNase contamination.

• DEPC-treated water (DEPC H ₂ O): Add 1 ml of diethylpyrocarbonate (DEPC) (0.1% final concentration) to 1 l of sterile water at 37°C overnight and then autoclave²

• 200 U/μl MMLV-RT,³ supplied with 5× MMLV-RT buffer and 0.1 M DTT (Invitrogen)

• Mix of 10 mM dNTPs: Add 80 μl of each 100 mM dNTP (dATP, dCTP, dGTP, and dTTP⁴) (Promega) to 480 μl DEPC H₂O and aliquot

• 100 ng/μl Pd(N) ₆ random hexamers (Amersham): Resuspend the lyophilized powder with DEPC H₂O to 1 μg/μl stock solution and then make aliquots of 100 ng/μl each

• 40 U/μl RNase inhibitor ⁵(Promega)

• 25 mM MgCl ₂ ⁶

2.2 Equipment

• Disinfected adjustable pipettes,⁷ range: 2–20 μl, 20–200 μl, 100–1,000 μl

• Nuclease-free aerosol-resistant pipette tips

• 1.5 and 0.2 ml tubes (autoclaved)

• Centrifuge suitable for centrifugation/spinning of 0.2 ml tubes at 13,200 or 14,000 rpm

• Thermal Cycler

2.3 Method

• 1. All RT assays should include a positive control (e.g., good-quality RNA from cell lines) and a negative one (DEPC H₂O or parallel extraction from a paraffin block without tissues). The amplifiability of genomic DNA (gDNA) in subsequent PCR should be ruled out performing a “no-RT” control, lacking the enzyme, or a control containing only gDNA.

  Add the following components to a 0.2 ml microcentrifuge tube, to a final volume of 7 μl:

  100 ng/μl Random hexamers	2.5–3 μl (250–300 ng final)
  Total RNA	1 ng–5 μg8_19, 9_19, 10_19
  DEPC H2O	to 7.05 μl

• 2. Heat the mixture of RNA, random primers, and water in a thermal cycler at 65°C for 10′ and quick chill on ice.

• 3. Add the following components to the mastermix, to a final volume of 20 μl:

  5× MMLV-RT buffer	4 μl (1× final)
  10 mM dNTPs	2 μl (1 mM final)
  0.1 M DTT	2 μl (10 mM final)
  40 U/μl RNase inhibitor	0.1 μl (4 units final)
  200 U/μl MMLV-RT	1.25 μl (250 units final)
  25 mM MgCl2	3.6 μl (7.5 mM final, considering the amount contained in the buffer)

• 4. Use the following Thermal Cycler programme:

  • 1×: 25°C for 10′

  • 1×: 37°C for 60′

  • 1×: 70°C for 15′

  • 1×: hold at 4°C

• 5. The cDNA can now be used as a template for PCR amplification. Store at –20°C until use. Avoid repeated freeze-thawing. The volume of first-strand reaction mixture to be used in the PCR should not exceed 10% of the amplification mixture volume, since larger amounts may result in decreased PCR product.

3 Reverse Transcription with AMV-RT and Specific Reverse Primer

It is possible to prime the RT using an antisense ­oligonucleotide, designed to target a specific mRNA transcript. Since specific primers’ melting temperature is higher than that of random oligos, the use of a thermostable enzyme, such as AMV (Avian Myelo­blastosis Virus Reverse Transcriptase) or Superscript is advisable. This protocol uses the AMV reverse transcriptase associated with a specific reverse primer. Target-specific primers synthesize the most specific cDNA and provide the most sensitive method of quantification [12], so it is the method of choice for the detection of scarcely expressed genes. The main disadvantage is that this approach requires separate priming reactions for each target gene, which is wasteful if only limited amounts of RNA are available. The use of more than one specific reverse primer in a single reaction tube (multiplex) is possible, but it requires careful experimental design and optimization of reaction conditions.

3.1 Reagents

Reagents from specific companies are reported here, but reagents of equal quality purchased from other companies may be used.

All reagents should be RNase free¹¹ or DEPC treated

• DEPC-treated water (DEPC H ₂ O): add 1 ml of diethylpyrocarbonate (DEPC) (0.1% final concentration) to 1 l of sterile water at 37°C overnight and then autoclave¹²

• 10 U/μl AMV-RT, supplied with 5× AMV-RT buffer (Promega)

• Mix of 10 mM dNTPs (Promega): add 80 μl of each 100 mM dNTP (dATP, dCTP, dGTP, and dTTP)¹³ to 480 μl DEPC H₂O and aliquot

• 30 pmol/ul specific reverse primer, ¹⁴: resuspend the lyophilized powder with DEPC H₂O to 300 pmol/μl stock solution and then make diluted aliquots of 30 pmol/μl each

• 40 U/μl RNase inhibitor ¹⁵ (Promega)

3.2 Equipment

• Disinfected adjustable pipettes,¹⁶ range: 2–20 μl, 20–200 μl, 100–1,000 μl

• Nuclease-free aerosol-resistant pipette tips

• 1.5 and 0.2 ml tubes (autoclaved)

• Centrifuge suitable for centrifugation/spinning of 0.2 ml tubes at 13,200 or 14,000 rpm

• Thermal Cycler

3.3 Method

• 1. All RT assays should include a positive control (e.g., good-quality RNA) and a negative one (DEPC H₂O). The amplifiability of gDNA in subsequent PCR should be ruled out performing a “no-RT” control, lacking the enzyme, or a control containing only gDNA.

  Add the following components to a 0.2 ml microcentrifuge tube, to a final volume of 10 microliters:

  5× AMV-RT buffer	2 μl (1× final)
  10 mM dNTPs	1 μl (1 mM final)
  30 pmol/μl Specific reverse primer	0.5 μl (15 pmol final)
  10 U/μl AMV-RT	0.2 μl (2 units final)
  40 U/μl RNase inhibitor	0.1 μl (4 units final)
  DEPC H2O	to 10 μl
  Total RNA	1 ng–2 μg17_19, 18_19, 19_19

• 2. Use the following Thermal Cycler programme:

  • 1×: 42°C for 60′

  • 1×: hold at 4°C

• 3. The cDNA can now be used as a template for amplification in PCR. The whole reverse transcription product can be used, provided that amplification reaction is performed in 50 μl in order to avoid inhibition of PCR. In such conditions, a PCR buffer without MgCl₂ is required.

3.4 Troubleshooting

Possible troubleshooting in the reverse transcription step is detected after PCR amplification (in this context, an optimized PCR assay is assumed). Refer to Table 19.1 for the most common problems and ­solutions concerning the cDNA synthesis step.

Table 19.1 Problems and solutions with the RT reaction