Reverse Transcription

All normal cells in a human's body, with few exceptions, have the same chromosomal DNA sequence, that is, the same genetic code. Thus, genomic information obtained from the DNA of easily obtained normal white blood cells would be applicable to the genetic makeup of normal lung, brain, or colon cells. This idea does not apply to malignant cells, which can have a genetic composition that is profoundly different from that of normal cells.

Function and structure of various cell types differ because of the mRNA that they transcribe and ultimately the proteins that are translated. In other words, it is the protein expression profile of cells that differentiates them. Presently, the most practical way to study the specific genes expressed in a particular cell type is to analyze the mRNA the cells make.

Because RNA is unstable and therefore difficult to work with in the laboratory, it can be converted into the complementary DNA (cDNA) by a process known as reverse transcrip-tion.69-71 The resultant cDNA is much more stable than the mRNA. Reverse transcription is so named because RNA is used as the template to direct the production of DNA: the reverse of normal cellular transcription, where DNA is used by RNA polymerase to direct the production of mRNA.

A reverse transcriptase enzyme is an RNA-directed DNA polymerase made and used by some RNA viruses to complete their life cycle within a host. Viral reverse transcriptases have been characterized and/or cloned, and the enzymes are commercially available for use in research and clinical molecular laboratories.72-76

Reverse transcriptase, similar to DNA polymerase, requires a DNA primer (Fig. 9.5) to initiate its function. Because mRNA has a poly(A) tail at its 3' 'end, an ideal primer for reverse transcription of mRNA species would be a poly(T) oligonucleotide (oligo dT).77,78 A replete collection of short DNA primers with random sequences can also be used; these primers are recommended if reverse transcription of ribosomal RNA (rRNA) is also desired along with mRNA. The enzyme starts transcription at the 3'-end of template RNA [the 5' -end of the nascent (new) cDNA strand] and proceeds in a 5'—> 3'-direction on the nascent strand ("first strand synthesis"). In this fashion, all the mRNA (or total RNA) present in a cell can be transcribed into complementary DNA. Those mRNA sequences that are present at a high copy number in the cell will be reverse transcribed to a high cDNA copy number compared with those mRNA sequences which are rare in a cell.

A typical reverse transcription protocol is given in Table 9.3. The two most commonly used reverse transcriptases are from bird and mouse viruses: avian myeloblastosis virus

Reverse Transcriptase
  1. 9.5. Reverse transcription. Reverse transcriptase uses oligo dT as the primer on the target mRNA and polymerizes in the 5'—> 3'-direction on the new DNA strand. The original mRNA strand is then cleaved by an RNAse domain within the reverse transcriptase (not shown), thus allowing polymerization of the single-stranded DNA into double-stranded DNA during poly-merase chain reaction .
  2. 9.5. Reverse transcription. Reverse transcriptase uses oligo dT as the primer on the target mRNA and polymerizes in the 5'—> 3'-direction on the new DNA strand. The original mRNA strand is then cleaved by an RNAse domain within the reverse transcriptase (not shown), thus allowing polymerization of the single-stranded DNA into double-stranded DNA during poly-merase chain reaction .

Table 9.3. Typical reverse transcription reaction protocol.

RNA 1-2 mg DEPC-treated H2O

Oligo dT or random primers at 40 mM 70°C I 5min 4°C I 5min

Quickly add 20.0 ml of prepared RT master mix

DEPC-treated H2O: add to total final volume

MMLV (10x) or AMV (5x) buffer dNTP at 10 mmole

RNAse inhibitor 10-40 units

MMLV reverse transcriptase 200 unitsa

AMV reverse transcriptase 30 unitsa

Use ~1-3 ml in the PCR reaction

  1. 0 ml 8.5 ml 2.0 ml
  2. 0 ml 2.0-4.0 ml 3.0 ml 1.0 ml

AMV avian myeloblastosis virus; DEPC diethylpyrocarbonate; dNTP deoxynucleotide triphosphate; MMLV Moloney murine leukemia virus; PCR polymerase chain reaction; RT reverse transcription aOne unit of reverse transcriptase is defined as the amount of enzyme that will incorporate 1 nmole of deoxythymidine triphosphate into acid-insoluble material in 10 min at 37°C using poly(rA), oligo(dT) as template primer

(AMV) and Moloney murine leukemia virus (MMLV).79 Their recommended buffers should not be interchanged. RNAse inhibitors and diethylpyrocarbonate-treated water are needed to preserve the unstable RNA. The initial 70°C heating is to remove secondary structures from the RNA; the 42°C (AMV and some MMLV products) or 37°C (some MMLV products) incubations are the working temperature of the enzymes. The 90-95°C step is needed to inactivate the enzymes.

The cDNA made by reverse transcription of mRNA (and/ or rRNA) can then be used as a template for PCR if the appropriate primers for the target DNA are present. During the first cycle of the PCR, only one (the forward) primer is needed because only one strand is polymerized, but this new strand will serve as the template for the opposite primer during the second PCR cycle, and polymerization of both strands will continue with each cycle. [Note that some bacteria such as Thermus thermophilus have an enzyme (Tth) that can both reverse transcribe RNA and polymerize DNA, allowing reverse transcription and PCR to proceed simultaneously in a single tube.]

Reverse transcription-PCR (RT-PCR) is thus an important tool that allows the investigator to study the genes expressed or not expressed in specific cells after isolation of the mRNA.80-83 Additional (post-PCR) techniques such as gel electrophoresis, single-stranded conformation polymorphism gels, restriction fragment length polymorphism analysis, DNA sequencing, microarrays, and so forth, can be applied to also determine if the genes expressed have mutations.

Under- or overexpression of a particular gene in neoplastic or reactive cells can be investigated by comparing their expression levels in normal cells, which could be done by comparing band strengths on Northern (RNA) blots. However, in these methods one must control for the number of tumor/reactive cells being the same as the number of normal cells. Analysis is much easier if done by real-time PCR, in which the ratio of the expression level of the gene of interest is compared with the expression level of a constitutively expressed housekeeping gene such as ß-actin, 18S rRNA, cyclophilin, glyceraldehyde-3-phosphate dehydrogenase, and ß2-microglobulin. This ratio is calculated in both the normal and neoplastic/reactive cells, and then the ratios are compared to see if there is relative up-or downregulation of the gene of interest.


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