Reverse-Transcription PCR to Bacterial Diagnostics
3.4. Data Analysis
For quantification analysis, design standard curves to span the expected range of RNA copy numbers. Include in each experiment dilutions correspond- ing to RNA copy number from 1 × 103 to 1 ⫻ 107. Reverse transcribe unknown
Fig. 2. Measurement of amplification efficiencies of standard curves and unknowns.
Shown in (A) are amplification curves from a serial dilution of in vitro-transcribed RNA templates. (B) shows the plot of the Ct values of the standard curve dilution series and the associated slope (s) and reaction efficiency (e). (Continued on next page) RNA corresponding to known cell equivalents and amplify by real-time PCR in parallel with the standards.
1. Integrated LightCycler data analysis software is used to plot a graph of the Ct values of both standard curve and dilutions of total RNA.
2. The slope of this line(s) can be used to calculate the amplification efficiency of the reactions (e) by application of the equation e = 10–1/S (see Fig. 2).
3. Amplification efficiencies of standards and unknowns must be as close as pos- sible for accurate quantification data to be returned.
Fig. 2. (Continued from previous page) (C) and (D) illustrate the amplification curves and plot of a dilution series prepared from total RNA. Amplification efficien- cies of both sets of templates are equivalent, meaning that interpolation of copy num- ber from these sets of amplifications can be performed in confidence.
4. Notes
1. RNA standards are prepared by in vitro transcription from a T7 promoter that has been attached to the 5´ end of the target gene. This generates a homogenous popu- lation of RNA molecules of known length. This material is quantified and the number molecules/microliter calculated. Serial dilutions of this RNA provide the template for the generation of an RT-PCR standard curve.
2. Any RT-PCR experiment that uses dilutions of RNA as starting material also will be influenced by inhibition of the RT reaction. The alternative approach, preparing a large stock of cDNA and diluting down from, this will not display this problem. There may be inhibition in the single large cDNA reaction but, because the standard curve is not designed to measure RT efficiency, just the subsequent PCR, its effect remains unnoticed. Amplification efficiencies are calculated by measuring the slope of a plot of Ct values for a dilution series of template (7), e = 10–1/S.
3. Short (~200 bp) PCR products from the genes of interest are cloned into plasmids containing a T7 RNA polymerase promoter site. In vitro transcription is used to generate RNA transcripts consisting of the cloned fragment and a short plasmid sequence. These transcripts are then column purified and quantified by ultravio- let spectroscopy. One 200-base RNA transcript is calculated to be equivalent to 0.2 ag (1 ag = 1 ⫻ 10–18 g) of this RNA. Serial dilutions of the in vitro-transcribed RNA are used as template for RT, and this cDNA is amplified using the Light- Cycler.
4. An alternative approach to generate starting template for in vitro transcription involves using PCR where the forward primer has been modified to include the T7 promoter sequence (taatacgactcactatagg). We chose the cloning strategy described here because it generates a stable pool of material from which fresh template can be prepared.
5. T7 RNA polymerase is highly processive and will continue transcription until a stop signal is encountered. In the case of a circular plasmid, the polymerase could potentially continue indefinitely. The restriction digestion prevents this and also results in the RNA transcripts being of uniform known size, which eases quanti- fication calculations.
6. Several methodologies suggest purification of the in vitro-transcribed RNA immedi- ately after transcription. However, it is our experience that it is advisable to perform DNase treatment of the prepared RNA before column purification. This step not only reduces the overall number of purification steps by one (it is no longer neces- sary to conduct a separate post-DNase purification) but it also facilitates the column purification of both free nucleotides and ribonucleotides from the mix in one step.
Column purification after DNase treatment also has been shown to improve the accuracy of spectrographic readings taken to quantify nucleic acid concentration.
7. Several online calculators are available to determine the molecular weight of an RNA transcript. A 200-base RNA will have an average molecular weight of 64 kDa, which corresponds to a single 200-base transcript having a mass of 0.1ag or 1 ⫻ 10–19g
8. Because of the large numbers of plates that will be involved, it is recommended that an initial “ranging” experiment be carried out to identify which dilutions will result in countable plates. Prepare 10-fold dilutions and plate 200 mL onto plate.
Incubate overnight at 37°C. Examine the plates to identify which of the dilutions result in between 30 and 300 CFU/plate. These are the dilutions that should be used in the triplicate plate count experiment.
9. The figures generated for amount of RNA per cell are limited by several factors.
First, the CFU per milliliter may underestimate the total number of cells present in the original culture as not all cell produce a colony. Second, no RNA prepara- tion method can be guaranteed to result in 100% purification of RNA from cul- tures. Therefore, we apply the term “recoverable RNA yield CFU–1” to more accurately describe our starting material.
10. At low concentrations of template, RT inhibits subsequent PCRs. The inhibitorary effect of RT on PCR is removed at template concentrations beyond 105 copies (14). When designing standard curves, it is useful to avoid lower dilutions of template because the amplification efficiency calculations will be affected by this inhibitory effect. An alternative solution mentioned (see Note 11) also may be useful.
11. We also have investigated the possibility of using a dilution series of cDNA samples to generate the standard curves. Provided that the amplification efficien- cies of these samples match those prepared from the RNA dilution series, this method provides several advantages. First, the total number of RT reactions is reduced; second, the cDNA template used to prepare the standards is more stable than an in vitro-transcribed RNA; third, this method is more accurate at lower template dilutions because it eliminates an inhibitory effect of components of the RT reaction on the PCR amplification. We found that diluting the high copy num- ber cDNA gave more consistent standard curves and a lower limit of detection (down to 100 copies) while having a similar amplification efficiency as the more laborious RNA dilution method.
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From: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O‘Connor © Humana Press Inc., Totowa, NJ