scholarly journals Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR

1999 ◽  
Vol 337 (2) ◽  
pp. 231-241 ◽  
Author(s):  
Junlong ZHANG ◽  
Christopher D. BYRNE
Keyword(s):  
Reverse Transcriptase ◽  
Copy Number ◽  
Accurate Determination ◽  
Fold Increase ◽  
Pcr Primers ◽  
Cdna Synthesis ◽  
Reverse Transcriptase Pcr ◽  
Target Mrna ◽  
Mrna Copy Number ◽  
Synthesis Reactions

Quantitative competitive reverse-transcriptase PCR is the most sensitive method for studying gene expression. To investigate whether the accuracy of the calculated target mRNA copy number is affected by the cDNA priming process, we utilized primers of different lengths, concentrations and primer sequences to prime cDNA synthesis reactions. Our results show a ≈ 19-fold increase in the calculated mRNA copy number from cDNA synthesis reactions primed with random hexamers (P< 0.001, n = 4), and a ≈ 4-fold increase in copy number with a specific hexamer (P< 0.001, n = 4) compared with that obtained with a 22-mer-sequence-specific primer. The increase in calculated mRNA copy number obtained by priming cDNA synthesis with the shorter specific and non-specific primers could be explained largely by the synthesis of truncated standard cDNA molecules lacking a requisite binding site for amplification with PCR primers. Since these truncated standard cDNA molecules could not be amplified and standard RNA is used to quantify target mRNA copy number, this phenomenon resulted in overestimation of target mRNA copy number. In conclusion, accurate determination of target mRNA copy number is most likely if a long specific antisense primer is used to prime cDNA synthesis.

scholarly journals Two variants of quantitative reverse transcriptase PCR used to show differential expression of α-, β- and γ-fibrinogen genes in rat liver lobes

1997 ◽  
Vol 321 (3) ◽  
pp. 769-776 ◽  
Author(s):  
Junlong ZHANG ◽  
Mina DESAI ◽  
Susan E. OZANNE ◽  
Cora DOHERTY ◽  
C. Nicholas HALES ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Rat Liver ◽  
Coefficient Of Variation ◽  
Copy Number ◽  
Internal Standard ◽  
Rt Pcr ◽  
Total Rna ◽  
Reverse Transcriptase Pcr ◽  
Mrna Copy Number ◽  
Sensitive Method

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of α-, β- and γ-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (α-fibrinogen F = 14.64, P = 0.0003; β-fibrinogen F = 3.74, P = 0.04; γ-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.

scholarly journals Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcriptase PCR

2001 ◽  
Vol 67 (5) ◽  
pp. 2384-2387 ◽  
Author(s):  
Mongi Benjeddou ◽  
Neil Leat ◽  
Mike Allsopp ◽  
Sean Davison
Keyword(s):  
Reverse Transcriptase ◽  
Pcr Primers ◽  
Rt Pcr ◽  
Black Queen Cell Virus ◽  
Silica Membrane ◽  
Blind Test ◽  
Reverse Transcriptase Pcr ◽  
Queen Cell ◽  
Acute Bee Paralysis Virus ◽  
Paralysis Virus

ABSTRACT A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3′ end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.

scholarly journals fbpABC Gene Cluster in Neisseria meningitidis Is Transcribed as an Operon

2000 ◽  
Vol 68 (12) ◽  
pp. 7166-7171 ◽  
Author(s):  
Heng H. Khun ◽  
Vinay Deved ◽  
Howard Wong ◽  
B. Craig Lee
Keyword(s):  
Reverse Transcriptase ◽  
Neisseria Meningitidis ◽  
Real Time ◽  
Gene Cluster ◽  
Quantitative Pcr ◽  
Pcr Amplification ◽  
Transcriptional Unit ◽  
Cdna Synthesis ◽  
Reverse Transcriptase Pcr ◽  
Real Time Quantitative Pcr

ABSTRACT The neisserial fbpABC locus has been proposed to constitute a single transcriptional unit. To confirm this operonic arrangement, transcription assays using reverse transcriptase PCR amplification were conducted with Neisseria meningitidis. The presence of fbpAB and fbpBC transcripts obtained by priming cDNA synthesis with anfbpC-sequence-specific oligonucleotide indicates thatfbpABC is organized as a single expression unit. The ratio of fbpA to fbpABC mRNA was approximately between 10- to 20-fold, as determined by real-time quantitative PCR.

scholarly journals Multiplex Quantitative Real-Time Reverse Transcriptase PCR for F+-Specific RNA Coliphages: a Method for Use in Microbial Source Tracking

2006 ◽  
Vol 73 (3) ◽  
pp. 808-814 ◽  
Author(s):  
Marek Kirs ◽  
David C. Smith
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Microbial Contamination ◽  
Accurate Determination ◽  
Microbial Source Tracking ◽  
Source Tracking ◽  
Rt Pcr ◽  
The Real ◽  
Reverse Transcriptase Pcr ◽  
Remediation Strategies

ABSTRACT It is well documented that microbial contamination of coastal waters poses a significant risk to human health through recreational exposure and consumption of shellfish. Identifying the source of microbial contamination (microbial source tracking) plays a dominant role in enabling effective management and remediation strategies. One method used to determine the source of the contamination is quantification of the ratio of the four subgroups of F+-specific RNA coliphages (family Leviviridae) in impacted water samples. Because of typically low concentrations in the environment, enrichment assays are performed prior to detection, even though differential replication rates have been reported. These assays are also compromised by differential loss of phage infectivity among subgroups after release into the environment, thus obscuring the initial ratio. Here, a culture-independent multiplex real-time reverse transcriptase-PCR (RT-PCR) protocol for the simultaneous quantification of all four subgroups of F+-specific RNA coliphages using novel primer sets and molecular beacons is presented. This assay is extremely sensitive, achieving detection with as few as 10 copies of isolated coliphage RNA, and is linear for a minimum of six orders of magnitude. During survival experiments, the real-time RT-PCR technique was able to quantify coliphages in seawater when culture-based double agar layer assay failed. While infectivity was lost at different rates at the subgroup level, decay constants in seawater, calculated using the real-time RT-PCR estimates, did not vary among subgroups. The accurate determination of the in situ concentration of F+-specific RNA coliphages using this method will facilitate more effective remediation strategies for impacted environments.

scholarly journals Systematical Engineering of Synthetic Yeast for Enhanced Production of Lycopene

2021 ◽  
Vol 8 (1) ◽  
pp. 14
Author(s):  
Yu Zhang ◽  
Tsan-Yu Chiu ◽  
Jin-Tao Zhang ◽  
Shu-Jie Wang ◽  
Shu-Wen Wang ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosome Rearrangement ◽  
Fold Increase ◽  
Host Strain ◽  
Target Product ◽  
Biosynthesis Pathway ◽  
Suitable Host ◽  
Enhanced Production ◽  
Condition Optimization ◽  
Heterologous Pathway

Synthetic biology allows the re-engineering of biological systems and promotes the development of bioengineering to a whole new level, showing great potential in biomanufacturing. Here, in order to make the heterologous lycopene biosynthesis pathway compatible with the host strain YSy 200, we evolved YSy200 using a unique Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE) system that is built in the Sc2.0 synthetic yeast. By inducing SCRaMbLE, we successfully identified a host strain YSy201 that can be served as a suitable host to maintain the heterologous lycopene biosynthesis pathway. Then, we optimized the lycopene biosynthesis pathway and further integrated into the rDNA arrays of YSy201 to increase its copy number. In combination with culturing condition optimization, we successfully screened out the final yeast strain YSy222, which showed a 129.5-fold increase of lycopene yield in comparison with its parental strain. Our work shows that, the strategy of combining the engineering efforts on both the lycopene biosynthesis pathway and the host strain can improve the compatibility between the heterologous pathway and the host strain, which can further effectively increase the yield of the target product.

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