Reverse Transcription of the Ribonucleic Acid:

The First Step in RT-PCR Assay

 Reverse transcriptase was discovered in 1970, and since then, it has played an instrumental role in the
advancement of molecular biology and biotechnology research. In the presence of all four deoxynucleotides
(dNTP: dATP, dCTP, dGTP, and dTTP) and under well-defined salt and pH conditions, the reverse transcriptase
extends a primer complementary to the RNA to produce a complementary DNA (cDNA) for the RNA template.
 is called “reverse transcription” because it reverses the flow of genetic information (from RNA to DNA, rather
than from DNA to RNA found in normal transcription).
 Oligonucleotide primers (oligo dT) or random primers are annealed to the RNA template and extended by the
reverse transcriptase in the presence of deoxynucleotides (dNTP) to make cDNA. RNAse H treatment can be used
to degrade RNA from RNA/DNA hybrids; however, the RNase H treatment is not required before standard PCR.
The denaturing step before PCR annealing and extension will make the cDNA available for the PCR primers and
Taq polymerase extension.
 The native reverse transcriptase is a multifunctional enzyme. In addition to its RNA-dependent DNA polymerase
activity, it also possesses both (1) a weak DNA-dependent DNA polymerase activity that is lacking the 3¢→5¢
exonuclease activity and (2) an RNase H activity that degrades the RNA template from RNA/ DNA hybrid strands
as the cDNA synthesis proceeds.

RT-PCR IN CHRONIC MYELOGENOUS LEUKEMIA:

 These characteristics have made it the method of choice for minimal residual disease monitoring such as in
chronic myelogenous leukemia (CML). CML comprises approximately 20% of all leukemias and is characterized by
a balanced (9;22) chromosomal translocation that results in the formation of a chimeric gene comprised of the
BCR (breakpoint cluster region) gene and the ABL oncogene (BCR-ABL fusion gene). The chimeric gene encodes a
fusion protein with constitutively increased tyrosine kinase activity, resulting in growth factor-independent
proliferation. This kinase is the target for current CML therapy, and BCR-ABL fusion gene levels are monitored to
determine the effectiveness of this therapy. This chapter uses BCRABL transcript detection to illustrate an
example for the RQ-PCR and describes a RQ-PCR method to detect the most common form of the BCR-ABL fusion
transcript in CML, known as p210 BCR-ABL.

Poly(A) PCR:
 Poly(A) PCR coordinately amplifies cDNA copies of all polyadenylated mRNAs, thereby generating a PCR product
(polyA cDNA) whose composition reflects the relative abundance of all expressed genes in the starting sample.
Poly(A) PCR enables global mRNA amplification from picogram amounts of RNA and has been routinely used to
analyse expression in small samples including single cells. The poly(A) cDNA pool generated is also indefinitely
renewable and as such represents a “molecular block”. Real-time PCR measurement, using gene-specific primers
and probes, of the expression levels of specific Indicator genes then allows gene signatures to be detected within
the poly(A) cDNA, thereby enabling expression profiling of very small amounts of starting material.

Single Cell RT-PCR on Mouse Embryos: A General Approach for Developmental Biology
Abstract:
Preimplantation development is a complicated process, which involves many genes. We have investigated the
expression patterns of 17 developmentally important genes and isoforms in early mouse embryos as well as in single cells
of the mouse embryo. The comparison is an excellent example for showing the importance of studying heterogeneity
among cell populations on the RNA level, which is being increasingly addressed in basic research and medical sciences,
particularly with a link to diagnostics (e.g. the analysis of circulating tumor cells and their progenitors). The ubiquitously
expressed histone variant H3f3a and the transcription factor Pou5f1 generated mRNA-derived products in all analyzed
preimplantation embryos (up to the morula stage) and in all analyzed blastomeres from 16-cell embryos, indicating a
rather uniform reactivation of pluripotency gene expression during mouse preimplantation development. In contrast,
genes that have been implicated in epigenetic genome reprogramming, such as DNA methyltransferases, methylcytosine-
binding proteins, or base excision repair genes revealed considerable variation between individual cells from the same
embryo and even higher variability between cells from different embryos. We conclude that at a given point of time, the
transcriptome encoding the reprogramming machinery and, by extrapolation, genome reprogramming differs between
blastomeres. It is tempting to speculate that cells expressing the reprogramming machinery have a higher developmental
potential.
RT PCR on AmpliGrid Chips: 1. Transfer either individual preimplantation embryos or individual blastomeres in
approximately 0.3ml of PBS each onto an AmpliGrid slide (Fig. 1c) (see Note 5). 2. Carry out reverse transcription with RT
primer mixture (Table 1) using a final primer concentration of 0.3mM each, using buffer and polymerases of the OneStep
RT PCR Kit according to the kit’s instructions (see Note 6). 3. Prepare a master mix as described above calculating
1.05×number of 1 ml reactions that are to be run on the AmpliGrid (see Note 7). 4. Add 1 ml of master mix to each
reaction spot (negative control, positive control, respective biological material such as single cell). 5. Cover each sample
with 5ml of covering solution to prevent evaporation. 6. Place the prepared slide on the AmpliSpeed Cycler. 7. Run the
appropriate thermal profile for reverse transcription on a preheated cycler, e.g., 60°C for 30 min. 8. Remove the 1ml
reaction volume (containing cDNA) from the chip, dilute 1:4 in double distilled H2 O and pipette 1ml onto empty spots of
an AmpliGrid. 9. Evaporate template cDNA to dryness on two different reaction sites of a second AmpliGrid (see Note 8).
10. Carry out two PCR reactions with the multiplex PCR kit and primer mixture 1 and 2 respectively (Table 1). Prepare a
master mix with all buffer substances, Taq polymerase and either PCR primer mix 1 or 2 at a final primer concentration of
0.3mM each according to the kit’s manual. Use the Q solution at 0.3-fold concentration. 11. Apply 1ml each of master
mix 1 and 2 to the two different reaction sites with the template and cover with 5ml of sealing solution. 12. Carry out PCR
on a slide cycler with an initial denaturation step of 95°C for 10 min and 40 cycles of 94°C for 30 s, 60°C for 60 s, and 72°C
for 60 s, and a final 10 min extension step at 72°C (see Note 9).
Troubleshooting:
 As in conventional tube-based reactions, the total volume of mastermix should be 5% more compared to the
exact calculated volume to ensure that the number of reactions can definitely be processed. This is especially
true for very low volumes like 1ml PCR, and we recommend to take into account a factor of 1.05×number of 1ml
reactions.
 Secondly, PCR conditions (time and temperatures during PCR) will influence the outcome dramatically. For a
technical evaluation of sensitivity, some of the genes of interest should be cloned into expression vectors and the
amount of synthetic gene sequences that can be detected should be known.