Pipett on ice and mix by pipetting gently up and down. Incubation of 60 min is recommended for cDNA fragments of more than 2, bp length. The optimal temperature depends on the structural features of the RNA. Note that optimal reaction time and temperature should be adjusted for each particular RNA.
Product Citations: Please click the black arrow on the right to expand the citation list. Click publication title for the full text. All reactions were split equally for two tubes. The first tube of each RT reaction was subjected to the PCI procedure followed by the EtOH precipitation, while the second one received no further treatment. The dilutions of RT reactions were made in a way that the amount corresponding to 1.
Each primer pair was implemented for three different RT reactions. All reactions were equally divided in two tubes after RT reaction. The first tube of each pair was subjected to the PCI procedure followed by EtOH precipitation, while the second one received no further treatment Figure 1.
We used two primer pairs for real-time PCR. These pairs were chosen for another reason: B2M gene expression level is moderate-high, and TBP is a relatively rarely expressed gene.
Finally, these transcripts are often considered as candidates for reference genes in the normalization procedure Figure 3 shows that the variability was dramatically decreased when the PCI procedure was implemented, especially for the first two points for 50 ng RT reactions. In general, C t values for the PCI sample are lower than those for the unpurified sample. It should also be mentioned that SD and CV of an unpurified sample with a B2M pair were improved with dilution points 4 and 5.
For TBP pairs the difference is also significant for the first two points with an increase of P -value for points 3 and 4. The high C t value variability of unpurified samples leads to poor reproducibility.
The ratio between three identical samples should be around 1 for every given point. This is the case when the PCI procedure was implemented. The ratio is in a range of 0. The lower C t values of the first point for samples without purification versus those for PCI samples were confirmed.
We thought that this effect could result from DNA synthesis activity of RT, so we tried to detect it using two approaches Figure 2. We decided to use a two-step approach, since the residual DNA synthesis activity of RT might be visible only when amplified with Taq Polymerase.
We did not observe any difference for a TBP pair. The average C t value for tubes with extra incubation was The control set for a B2M pair has a slightly lower C t value compared with that for tubes with extra incubation It is noteworthy that the same amount of reverse transcriptase was used in both—control tubes and tubes with extra incubations.
We were able to see more dramatic differences when the samples with RT were compared with samples without it. The C t value was The differences were statistically significant with corresponding P -values equal to 0. To check any influence of RT dilution buffer with NP on PCR, the control set of tubes was established with water substituted for the buffer.
Each tube of the pair was subjected to EtOH precipitation as already described, but a PCI procedure was implemented only for the second tube of the pair. Although, it has to be pointed out that the variability was decreased even for samples after EtOH precipitation alone, when compared with unpurified samples Supplementary Table 4 and Table 1. The samples after EtOH precipitation, when the PCI step was omitted, tend to exhibit artificially increased amplification efficiencies 2.
A yield of The experiments were also performed in order to evaluate template loss during the PCI procedure followed by EtOH precipitation. The RT reactions with LTP spikes were split into two tubes, extracted and precipitated as described above. The control tube with the same amount of LTP amplicon but without any further treatment was used as a reference.
The recovery percentage was The experiments were carried out on three RT reactions in one experiment and were repeated on two different days. Using this formula Equation 10 we may calculate C t values in every dilution point with every IP combination—IP in the first point plus any decrement. This example shows from theoretical a standpoint how more diluted samples may exhibit lower C t values than those for undiluted ones.
The derived amplification efficiencies and coefficients of correlation are presented in Table 3. Several tendencies were observed. The coefficients of correlation were increased when most concentrated points under the biggest inhibitory influence were excluded from the calibration curve. It is acknowledged that the efficiency calculation from the slope of a calibration curve often overestimates PCR efficiency We have observed this in our experiments, and it has been reported by others 8 , 15 , This seems a bit paradoxical, and likewise is the finding of an overestimated efficiency in samples containing PCR inhibitors.
Such a paradox could be reconciled by considering PCR competent molecules as an increasing percentage of the total molecules in a PCR tube with each sequential dilution. It seems that a key factor is reverse transcriptase itself 3 — 5 , in spite of minor potential influences by other RT reaction components 5 , 8. Reverse transcriptase may exhibit several activities: A previous study has suggested that RT inhibition of PCR is mediated through direct interaction with a specific primer-template combination 4.
RT molecules which were not bound to such a complexes may have dramatically reduced DNA synthesis capabilities, or no such an activity at all, while possessing the binding ability during post RT step manipulations.
RNase H activity, if the reverse transcriptase construct was not mutated to remove it;. We were unable to demonstrate DNA polymerase activity in an experiment where Taq Polymerase was substituted with RT, but if such activity is retained during a few cycles, the signal would be undetectable and possibly lost in background. The effects we observed in our experiments could be explained by differential residual DNA polymerase activity and binding capacity of two RT fractions, free and bound.
We believe there are two RT activities with opposite vectors: i RT amplifies the template that would lead to an overestimation of the number of molecules; and ii molecules of RT compete for binding sites with Taq Polymerase molecules, decreasing the amount of template to be detected. This ratio decreases with each sequential dilution, which also causes a decrease in inhibition. Still, the results were confirmed. No such effect was detected. When the first point of unpurified sample was excluded from the calibration curve, the linearity was improved for both pairs, B2M and TBP, while there was no effect on linearity by the same approach for the PCI sample.
These facts allow us to assume that the overestimation of number of molecules in the first point could be ascribed to an effect of unpurified sample, and it was probably caused by DNA polymerase activity of RT. Even though the IP should be highest at this point when compared with that of other dilution points, the DNA polymerase activity appears to be highest also. It could be the case that DNA synthesis activity surpasses the inhibition in this particular dilution point.
It means that 2—5 molecules of RT could be required to create a complementary strand of bp amplicon. In fact, this number could be even larger. This is predetermined by the nature of amplification with a pair of specific primers.
Only one-fourth of molecules after two PCR cycles have the target amplicon size, and other molecules are extended beyond the amplicon borders. To add a degree of complication to this interpretation, we may also assume that the binding affinities of free and bound RT fractions are different.
Moreover, the binding capacity of both fractions may decrease with unknown dynamics during PCR. Whether or not RT directly interacts with Taq Polymerase is also an open question 1 , 4.
The DNA synthesis activity and binding capacity of RT molecules may be influenced by numerous factors. Obviously, various specific pairs of primers taken for PCR will be under different levels of inhibition even within the same transcript, since the primer—template structure strongly influences RT binding affinity This could explain the different levels of inhibition seen for different sets of primers.
All of this argues strongly in favor for removing reverse transcriptase molecules before performing real-time PCR. The method of purification we present here could be considered to be a universal equalizer, regardless of what RT type and conditions are implemented before PCR.
We introduced the organic extraction step 34 before ethanol precipitation, because the proteins could be copurified if ethanol precipitation alone is utilized Implementation of the described approach leads to an improvement of real-time PCR results. Variability is decreased and the precision of detection is increased when a PCI procedure followed by ethanol precipitation is applied. When undiluted RT reactions starting with nanograms of total RNA are taken into PCR without purification, the inhibitory effects will be at their maxima.
To decrease these effects, an investigator usually dilutes the RT reaction before PCR, but the resulting C t values will be in an area with increased variability, low reproducibility and inconsistent detection 12 , 36 , Our approach allows for template concentration by eliminating PCR inhibitors, which would be especially valuable in the detection of rarely expressed transcripts, or in clinical and environmental screening applications.
It is reasonable to expect that reverse transcriptase may also affect other downstream applications, e. In this assay, supercoiled plasmid DNA was incubated with varying DTT concentrations and subsequently analyzed in an agarose gel as described in the materials and methods section.
The bands representing nicked plasmid were quantified and the results shown in Figure 1 A. Incubating the plasmid DNA with DTT resulted in a relatively weak, yet detectable, dose dependent increase in the intensity of the band representing nicked plasmid from arbitrary units in the absence of DTT to , , and when the plasmid was incubated with 0.
Interestingly, this effect was observed even without any added copper, which was previously thought necessary for thiol mediated nicking of DNA [ 11 , 15 , 16 ]. A Bar chart showing the results of incubating plasmid DNA with varying concentrations of DTT and separating the reaction products in an agarose gel. The chart shows the results of quantifying the bands representing nicked plasmid.
The results are shown as raw values arising from the quantification arbitrary units. To further elucidate the nature of the DTT induced DNA nicks, we set up a second nick-sensing experiment, a modified nick translation assay, which is schematically depicted in Figure 1 B. Subsequently, the DNA was bound to a nylon membrane and the amount of incorporated radiolabelling was visualized using a phosphorimager and quantified using ImageJ.
The results obtained using this modified nick translation assay are shown in Figure 1 C. The signal intensity arising from samples incubated without DTT was arbitrary units. The signal intensity rose upon incubation with 0. The samples incubated with 0.
A negative control incubated with 10 mM DTT but not incubated with Taq polymerase was included data not shown. In this sample, it was not possible to detect any radiolabelling on the membrane demonstrating that the assay is specific for detection of polymerase-mediated incorporation of radiolabelled nucleotides. In the present study, we focus on the consequences of thiol mediated nicking of DNA under typical experimental conditions.
For this reason, the presented experiments display two key differences from most of the previous literature on the DNA-cleaving activity of thiols, since these studies have mainly focused on elucidating the mechanisms behind the reaction pathway [ 9 , 16 , 23 , 24 , 25 ]. Firstly, our results were obtained without the deliberate addition of copper and secondly, the thiol concentrations used in this study range from 0.
Immobilization of DNA forms the foundation of many modern DNA sensor studies including fluorescence microscopic, graphene electronic, and plasmon resonance based methods which may be coupled with a polymerase amplification enabled signal amplification step [ 26 , 27 , 28 ]. The functionalized glass slides were incubated with fluorescently labelled bp PCR product either in the presence or absence of DTT.
Using fluorescence microscopy, the amount of DNA immobilized on the microscopy slide could be quantified Figure 2 A. The number of signals per image frame was quantified using ImageJ and the results shown in Figure 2 B. The results shown are the number of DNA molecules visible per image frame.
The number of DNA molecules bound to the surface was increased by more than a factor of two from an average of signals per image frame no DTT added to an average of signals per image frame upon addition of 10 mM DTT mean of six independent experiments , strongly suggesting that DTT stimulates immobilization of DNA to NHS-ester functionalized slides and certainly may influence results obtained using such surfaces.
A schematic depiction of this assay is shown in Figure 3 A. RCA in turn generates long tandem repeat products that can be visualized at the single molecule level by hybridization of specific fluorescent probes and the results obtained by counting the number of signals using a fluorescence microscope.
Both in the presence and the absence of IN, the number of signals per image frame rose with approximately the same number 19 and 18 respectively upon addition of DTT. DTT thus markedly increased the background, and thereby reduced the signal to noise ratio, creating serious problems for the assay readout.
A Schematic depiction of a novel method for detecting IN see results and discussion for details ; B The results of the assay outlined in A when performed either with the two rightmost columns or without the two leftmost columns IN. The assay was performed with or without addition of 10 mM DTT during the circle immobilization step as indicated on the graph.
Individual RCA products were visualized using fluorescence microscopy and their number was quantified using ImageJ. These results clearly highlight the importance of meticulously ensuring consistent reaction conditions as well as to be aware of the possibility that DTT may cause unintended side reactions altering the outcome of single molecule detecting experiments and other ultrasensitive assays.
Using standard molecular biological reaction conditions, we show that DTT is able to introduce single stranded nicks in double stranded DNA. Furthermore, DTT was able to facilitate immobilization of fluorescently labelled DNA to a functionalized microscopy slide. Although being too modest to substantially affect the results of traditional bulk assay setups, the effect of DTT was strong enough to severely affect the results of a recently developed single molecule detection REEAD setup, demonstrating a potential impact on single molecule detection protocols.
Unlike previous studies investigating thiol mediated DNA effects, the reported results were obtained using reaction conditions without any added copper.
Our findings underscore the importance of carefully ensuring uniformity of reaction conditions when performing single molecule studies and of being aware of buffer additives in general and DTT in particular.
In this assay, we utilized the fact that nicked DNA is separated from supercoiled and relaxed DNA when electrophoresed in the presence of the DNA intercalating dye ethidium bromide.
This is due to the fact that ethidium bromide binding introduces positive supercoiling in relaxed plasmid DNA, which results in an increased mobility. Nicked plasmid DNA retains the mobility of relaxed DNA even in the presence of ethidium bromide, since the introduced overwinding escapes via the nick. The high mobility bands represent intact plasmid DNA and the retarded bands represent nicked plasmid. National Center for Biotechnology Information , U. Journal List Sensors Basel v. Sensors Basel.
Published online May Find articles by Marie Bech Andersen. Find articles by Jonas Thomsen. Find articles by Jing Wang. Nicole Jaffrezic-Renault, Academic Editor. Author information Article notes Copyright and License information Disclaimer.
Received Mar 13; Accepted May
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