scholarly journals Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk

2013 ◽  
Vol 96 (12) ◽  
pp. 7932-7944 ◽  
Author(s):  
P. Modesto ◽  
S. Peletto ◽  
G. Pisoni ◽  
P. Cremonesi ◽  
B. Castiglioni ◽  
...  
Keyword(s):  
Real Time ◽  
Expression Analysis ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Somatic Cells ◽  
Goat Milk ◽  
Internal Reference ◽  
Quantitative Expression ◽  
Reverse Transcription Pcr

scholarly journals Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood

2011 ◽  
Vol 12 (11) ◽  
pp. 7732-7747 ◽  
Author(s):  
Simone Peletto ◽  
Simone Bertuzzi ◽  
Chiara Campanella ◽  
Paola Modesto ◽  
Maria Grazia Maniaci ◽  
...  
Keyword(s):  
Real Time ◽  
Expression Analysis ◽  
Real Time Pcr ◽  
Whole Blood ◽  
Reference Genes ◽  
Internal Reference ◽  
Quantitative Expression

scholarly journals Selection of internal references for transcriptomics and RT-qPCR assays in Neurofibromatosis type 1 (NF1) related Schwann cell lines

2020 ◽  
Author(s):  
Yi-Hui Gu ◽  
Xi-Wei Cui ◽  
Jie-Yi Ren ◽  
Man-Mei Long ◽  
Wei Wang ◽  
...  
Keyword(s):  
Schwann Cell ◽  
Real Time ◽  
Cell Lines ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Neurofibromatosis Type ◽  
Internal Reference ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Reference Selection

AbstractTranscriptomics has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the most qualified reference genes for the relative quantitation analysis of transcriptomics and real-time quantitative reverse-transcription PCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most used internal reference gene, was significantly unstable. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. Our recommended internal reference genes may improve the accuracy and reproducibility of the results of transcriptomics and real-time quantitative reverse-transcription PCR in further gene expression analyses of NF1 related tumors.

scholarly journals Simultaneous Absolute Quantification of Target and Control Templates by Real-Time Fluorescence Reverse Transcription-PCR Using 4-(4′-Dimethylaminophenylazo)benzoic Acid as a Dark Quencher Dye

2001 ◽  
Vol 47 (3) ◽  
pp. 486-490 ◽  
Author(s):  
Karl-Anton Kreuzer ◽  
Alexander Bohn ◽  
Joachim Lupberger ◽  
Jerome Solassol ◽  
Philipp le Coutre ◽  
...  
Keyword(s):  
Benzoic Acid ◽  
Real Time ◽  
Correlation Coefficient ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Philadelphia Chromosome ◽  
Myelogenous Leukemia ◽  
Rt Pcr ◽  
Internal Reference ◽  
Reverse Transcription Pcr

Abstract Background: Despite the many advantages of real-time fluorescence reverse transcription-PCR (RT-PCR) as a quantitative analytical tool, simultaneous quantification of target and reference templates within one reaction has not been reported. We developed such an assay with an internal reference template. Methods: For quantification of target and reference sequences, we used two fluorescent probes in one reaction vessel on an ABI PRISM 7700 SDS instrument. Fluorescent probes were labeled with either 6-carboxy-fluorescein or hexachloro-6-carboxy-fluorescein as reporter dye and 4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL) as a dark quencher fluorophore. To test the sensitivity and specificity of this assay, serial dilutions of reference and target templates were analyzed in one PCR reaction. In the presence of 10 β-actin molecules as control templates, 105 bcr/abl molecules were amplified, and 105 β-actin molecules were amplified in the presence of 10 bcr/abl copies. We also performed single and duplex measurements on samples from five patients with documented Philadelphia chromosome-positive chronic myelogenous leukemia disease courses (72 samples) and three with minor bcr/abl+ acute myelogenous leukemias (26 samples). Results: For M-bcr/abl duplex RT-PCR, the correlation coefficient (r) for starting template amounts and threshold cycle values was 0.99; for m-bcr/abl, r = 0.96, indicating a precise log-linear relation for 10–105 copies/100 ng of cDNA. In the same PCR reactions, r = 0.99 for β-actin (coamplified with M-bcr/abl or m-bcr/abl) for 103–107 copies/100 ng cDNA. The linear correlation coefficient for single and duplex measurements was 0.98 for M- and m-bcr/abl in patient samples. Conclusions: DABCYL can be used as dark quencher fluorophore in real-time fluorescence PCR. The duplex fluorescence RT-PCR assay for bcr/abl and β-actin transcripts allows monitoring of bcr/abl+ leukemias.

Sign in / Sign up

Export Citation Format

Share Document