Simple, sensitive and accurate method for the quantification of prothrombin mRNA by using competitive PCR

2001 ◽  
Vol 356 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Phulwinder K. GROVER ◽  
Alan M. F. STAPLETON ◽  
Katsuhito MIYAZAWA ◽  
Rosemary Lyons RYALL
Keyword(s):  
Reverse Transcription ◽  
Accurate Method ◽  
Competitive Pcr ◽  
Tissue Samples ◽  
Total Rna ◽  
Standard Curve ◽  
Minimum Number ◽  
Identical Nucleotide ◽  
Number Of Cycles ◽  
Made In

A method for the quantification of prothrombin (PT) mRNA species in hepatic tissues of rats was developed with the use of competitive PCR. To validate the quantification approach, sequential dilutions of total RNA from one of the samples were reverse transcribed. Their equivalent volumes were amplified together with a known amount of non-homologous competitor cDNA with identical nucleotide primers. The disparate sizes of target and competitor permitted the easy identification and quantification of bands in samples after densitometric analysis of ethidium bromide-stained agarose gels. Ratios of intensities of target and competitor bands were plotted against the initial amounts of total RNA species used, giving a linear relationship. The slope of this line was virtually identical with that obtained when the sample RNA was replaced with recombinant target cDNA, indicating that recombinant cDNA behaved in PCR identically with that made by reverse transcription and permitting the estimation of transcripts in reverse transcription reactions by using the recombinant counterpart of each as a standard. To avoid variation in the final results, the amount of competitor used in the assay was calculated separately from the equivalence point of the reverse-transcribed total RNA of one of the tissue samples; PCR was performed only for the minimum number of cycles required to detect products. A standard curve was made in each PCR run by amplifying differing amounts of recombinant cDNA species of PT or β-actin together with a constant amount of its competitor. The numbers of transcripts in the tissues were then determined directly by PCR incorporating the same amount of respective competitor (as used in the standard curve) and comparing the ratios of products with the standard curve. Application of this method revealed that the median ratio of PT message to β-actin message in hepatic tissues of 10 normal rats was 0.37, with a mean±S.D. of 0.37±0.07 (range 0.27–0.47). Although the method was developed for the quantification of PT transcripts in liver, it can easily be used for non-hepatic tissues as well. The technique is simple, quick and sensitive and requires only a very small amount of substrate.

scholarly journals Validation of Reference Genes for Normalization Gene Expression in Reverse Transcription Quantitative PCR in Human Normal Thyroid and Goiter Tissue

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Raquel Weber ◽  
Ana Paula Santin Bertoni ◽  
Laura Walter Bessestil ◽  
Beatriz Maria de Azevedo Assis Brasil ◽  
llma Simoni Brum ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Quantitative Polymerase Chain Reaction ◽  
Thyroid Tissue ◽  
Data Interpretation ◽  
Accurate Method ◽  
Tissue Samples ◽  
Normal Thyroid ◽  
Normalization Gene ◽  
The Stability ◽  
Beta 2 Microglobulin

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been recognized as the most accurate method for quantifying mRNA transcripts, but normalization of samples is a prerequisite for correct data interpretation. So, this study aimed to evaluate the most stable reference gene for RT-qPCR in human normal thyroid and goiter tissues. Beta-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); succinate dehydrogenase, subunit A, flavoprotein (Fp) (SDHA); hypoxanthine phosphoribosyltransferase I (HPRTI); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ); and beta-2-microglobulin (B2M) were evaluated in 14 thyroid tissue samples (7 normal and 7 goiter tissues) by RT-qPCR. The mean Cq and the maximum fold change (MFC) and NormFinder software were used to assess the stability of the genes. As a result, ACTB gene was more stable than GAPDH, SDHA, HPRTI, YWHAZ, and B2M. In conclusion, ACTB could be used to normalize RT-qPCR data in normal thyroid and goiter tissues.

scholarly journals A rapid and accurate method for quantitating total RNA transferred during Northern blot analysis

1988 ◽  
Vol 16 (5) ◽  
pp. 2354-2354 ◽  
Author(s):  
Nathalie Denis ◽  
Daniel Corcos ◽  
Jacques Kruh ◽  
Alain Kitzis
Keyword(s):  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Accurate Method ◽  
Total Rna

scholarly journals Downregulation and Prognostic Performance of MicroRNA 224 Expression in Prostate Cancer

2013 ◽  
Vol 59 (1) ◽  
pp. 261-269 ◽  
Author(s):  
Konstantinos Mavridis ◽  
Konstantinos Stravodimos ◽  
Andreas Scorilas
Keyword(s):  
Prostate Cancer ◽  
Cox Regression ◽  
Specific Antigen ◽  
Progression Free Survival ◽  
Prognostic Indicator ◽  
Disease Stage ◽  
Clinical Value ◽  
Prostate Tumors ◽  
Tissue Samples ◽  
Total Rna

INTRODUCTION The extensive use of prostate-specific antigen as a general prostate cancer biomarker has introduced the hazards of overdiagnosis and overtreatment. Recent studies have revealed the immense biomarker capacity of microRNAs (miRNAs) in prostate cancer. The aim of this study was to analyze the expression pattern of miR-224, a cancer-related miRNA, in prostate tumors and investigate its clinical utility. METHODS Total RNA was isolated from 139 prostate tissue samples. After the polyadenylation of total RNA by poly(A) polymerase, cDNA was synthesized with a suitable poly(T) adapter. miR-224 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method, Cq(2−ΔΔCq). We performed comprehensive biostatistical analyses to explore the clinical value of miR-224 in prostate cancer. RESULTS miR-224 expression was significantly downregulated in malignant samples compared with benign samples (P < 0.001). Higher miR-224 expression levels were found in prostate tumors that were less aggressive (P = 0.017) and in an earlier disease stage (P = 0.018). Patients with prostate cancer who were positive for miR-224 had significantly enhanced progression-free survival intervals compared with miR-224–negative patients (P = 0.021). Univariate bootstrap Cox regression confirmed that miR-224 was associated with favorable prognosis (hazard ratio 0.314, P = 0.013); nonetheless, multivariate analysis, adjusted for conventional markers, did not identify miR-224 as an independent prognostic indicator. CONCLUSIONS miR-224 is aberrantly expressed in prostate cancer. Its assessment by cost-effective quantitative molecular methodologies could provide a useful biomarker for prostate cancer.

High microRNA-28-5p expression in colorectal adenocarcinoma predicts short-term relapse of node-negative patients and poor overall survival of patients with non-metastatic disease

2018 ◽  
Vol 56 (6) ◽  
pp. 990-1000 ◽  
Author(s):  
Panagiotis Tsiakanikas ◽  
Christos K. Kontos ◽  
Dimitrios Kerimis ◽  
Iordanis N. Papadopoulos ◽  
Andreas Scorilas
Keyword(s):  
Overall Survival ◽  
Reverse Transcription ◽  
Colorectal Adenocarcinoma ◽  
Quantitative Polymerase Chain Reaction ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
Colorectal Tumors ◽  
Mesenchymal Transition ◽  
Cox Regression Analysis ◽  
Total Rna

Abstract Background: MicroRNAs (miRNAs) may function either as oncogenes or tumor suppressors and are heavily involved in the initiation and progression of cancer, and in metastasis of tumor cells. MicroRNA-28-5p (miR-28-5p) targets several cancer-related genes and is hence involved in cell proliferation, migration, invasion and epithelial-mesenchymal transition. In this study, we investigated the potential diagnostic and prognostic significance of miR-28-5p expression in colorectal adenocarcinoma, the most frequent type of colorectal cancer (CRC). Methods: Therefore, we isolated total RNA from 182 colorectal adenocarcinoma specimens and 86 paired non-cancerous colorectal mucosae. After polyadenylation of 2 μg total RNA and its reverse transcription using an oligo-dT-adapter primer, we quantified miR-28-5p levels using an in-house-developed reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR) method, based on the SYBR Green chemistry. Results: Comparison of miR-28-5p levels among 86 pairs of colorectal tumors and their adjacent non-cancerous mucosae uncovered the downregulation of miR-28-5p expression in the majority of malignant colorectal tumors. More importantly, high miR-28-5p expression predicts poor disease-free survival (DFS) and overall survival (OS) of colorectal adenocarcinoma patients. Multivariate Cox regression analysis revealed that miR-28-5p overexpression is a significant predictor of poor prognosis in colorectal adenocarcinoma, independent of tumor size, histological grade, TNM staging, radiotherapy and chemotherapy. Interestingly, strong miR-28-5p expression retains its predictive potential regarding relapse among patients with negative regional lymph nodes, and predicts poor OS in patients diagnosed with non-metastatic colorectal adenocarcinoma. Conclusions: High miR-28-5p expression predicts poor DFS and OS of colorectal adenocarcinoma patients, independently of clinicopathological prognosticators and standard patient treatment, including radiotherapy and chemotherapy.

scholarly journals Detection of Viable Cryptosporidium parvum in Soil by Reverse Transcription–Real-Time PCR Targetinghsp70mRNA

2011 ◽  
Vol 77 (18) ◽  
pp. 6476-6485 ◽  
Author(s):  
Zhanbei Liang ◽  
Ann Keeley
Keyword(s):  
Cryptosporidium Parvum ◽  
Reverse Transcription ◽  
Rna Binding ◽  
Direct Detection ◽  
Rna Extraction ◽  
Milk Powder ◽  
Extraction Methods ◽  
Content Type ◽  
Total Rna ◽  
Rna Extraction Methods

ABSTRACTExtraction of high-quality mRNA fromCryptosporidium parvumis a key step in PCR detection of viable oocysts in environmental samples. Current methods for monitoring oocysts are limited to water samples; therefore, the goal of this study was to develop a rapid and sensitive procedure forCryptosporidiumdetection in soil samples. The efficiencies of five RNA extraction methods were compared (mRNA extraction with the Dynabeads mRNA Direct kit after chemical and physical sample treatments, and total RNA extraction methods using the FastRNA Pro Soil-Direct, PowerSoil Total RNA, E.Z.N.A. soil RNA, and Norgen soil RNA purification kits) for the direct detection ofCryptosporidiumwith oocyst-spiked sandy, loamy, and clay soils by using TaqMan reverse transcription-PCR. The study also evaluated the presence of inhibitors by synthesis and incorporation of an internal positive control (IPC) RNA into reverse transcription amplifications, used different facilitators (bovine serum albumin, yeast RNA, salmon DNA, skim milk powder, casein, polyvinylpyrrolidone, sodium hexametaphosphate, andSalmonella entericaserovar Typhi) to mitigate RNA binding on soil components, and applied various treatments (β-mercaptoethanol and bead beating) to inactivate RNase and ensure the complete lysis of oocysts. The results of spiking studies showed thatSalmonellacells most efficiently relieved binding of RNA. With the inclusion ofSalmonelladuring extraction, the most efficient mRNA method was Dynabeads, with a detection limit of 6 × 102oocysts g−1of sandy soil. The most efficient total RNA method was PowerSoil, with detection limits of 1.5 × 102, 1.5 × 103, and 1.5 × 104C. parvumoocysts g−1soil for sandy, loamy, and clay samples, respectively.

Quantification of G protein mRNA using reverse transcription and competitive PCR with a colorimetric microplate assay

1998 ◽  
Vol 12 (1) ◽  
pp. 15-25 ◽  
Author(s):  
Rupa Mokkapatti ◽  
Richard A Conn ◽  
Joseph A Carcillo ◽  
Marjorie Romkes ◽  
Edwin K Jackson
Keyword(s):  
G Protein ◽  
Reverse Transcription ◽  
Microplate Assay ◽  
Competitive Pcr

scholarly journals Evaluation of the quality of RNA extracted from archival FFPE glioblastoma and epilepsy surgical samples for gene expression assays

2018 ◽  
Vol 71 (8) ◽  
pp. 695-701 ◽  
Author(s):  
Harry R Haynes ◽  
Clare L Killick-Cole ◽  
Kelly M Hares ◽  
Juliana Redondo ◽  
Kevin C Kemp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Total Rna ◽  
Expression Studies ◽  
Significant Difference ◽  
Gene Expression Analyses ◽  
Gene Expression Studies ◽  
And Control

AimsHistopathological tissue samples are being increasingly used as sources of nucleic acids in molecular pathology translational research. This study investigated the suitability of glioblastoma and control central nervous system (CNS) formalin-fixed paraffin embedded (FFPE) tissue-derived RNA for gene expression analyses.MethodsTotal RNA was extracted from control (temporal lobe resection tissue) and glioblastoma FFPE tissue samples. RNA purity (260/280 ratios) was determined and RNA integrity number (RIN) analysis was performed. RNA was subsequently used for RT-qPCR for two reference genes,18SandGAPDH.ResultsReference gene expression was equivalent between control and glioblastoma tissue when using RNA extracted from FFPE tissue, which has key implications for biological normalisation for CNS gene expression studies. There was a significant difference between the mean RIN values of control and glioblastoma FFPE tissue. There was no significant correlation between 260/280 or RIN values versus total RNA yield. The age of the tissue blocks did not influence RNA yield, fragmentation or purity. There was no significant correlation between RIN or 260/280 ratios and mean qPCR cycle threshold for either reference gene.ConclusionsThis study showed that routinely available CNS FFPE tissue is suitable for RNA extraction and downstream gene expression studies, even after 60 months of storage. Substantial RNA fragmentation associated with glioblastoma and control FFPE tissue blocks did not preclude downstream RT-qPCR gene expression analyses. Cross validation with both archival and prospectively collated FFPE specimens is required to further demonstrate that CNS tissue blocks can be used in novel translational molecular biomarker studies.

169 AN OPTIMIZED PROTOCOL FOR EXTRACTING RNA FROM SINGLE BOVINE OOCYTES AND BLASTOMERES

2008 ◽  
Vol 20 (1) ◽  
pp. 164 ◽  
Author(s):  
M. Boelhauve ◽  
T. Guengoer ◽  
K. Zitta ◽  
V. Zakhartchenko ◽  
E. Wolf
Keyword(s):  
Liquid Nitrogen ◽  
Reverse Transcription ◽  
Magnetic Beads ◽  
Isolation Method ◽  
Total Rna ◽  
Qpcr Analysis ◽  
Optimized Protocol ◽  
Single Blastomeres ◽  
Bovine Oocytes ◽  
Rna Concentration

Quantitative PCR (qPCR) analysis of gene expression in single bovine oocytes or blastomeres from early preimplantation embryos needs to meet optimized requirements for the isolation of the RNA, the reverse transcription reaction, and even the qPCR. Normally, large amounts of tissues are required for extracting RNA, but the RNA recovery per embryo is too low for a reliable detection of low expressed genes. Therefore an optimized isolation method is essential for obtaining sufficient RNA recoveries of (a) a group of embryos/oocytes (n = 10), (b) a single oocyte/embryo, or (c) single blastomeres without inhibition of the subsequent steps. For each experiment studied, the RNA was reverse-transcribed with ExtremeScript-OLS� (Omni Life Science, Inc., Raynham, MA, USA) following the manufacturer's instructions. The transcript levels were analyzed by the detection of the genes STAT3 and LEPR. First, we compared different isolation protocols from the literature for the isolation of oocyte RNA[TriZol� (Invitrogen, Carlsbad, CA, USA) and RNAPure™ (Peqlab Biotechnologie, Erlangen, Germany) for isolation of total RNA; magnetic beads and Absolutely RNA™ Nanoprep (Stratogene, La Jolla, CA, USA) for mRNA; n = 12 per protocol] by measurement of the total RNA concentration (TriZol and RNAPure) and qPCR analysis (all isolation techniques). The results showed that RNAPure provided the highest transcript numbers (100% RNAPure v. 78% TriZol, 25% Nanoprep, and 5% magnetic beads, respectively; P ≤ 0.001) and also the highest total RNA concentration (2.1 ng µL–1 v. TriZol 1.5 ng µL–1 total RNA per oocyte; P ≤ 0.05). In the second experiment, we analyzed the influence of a coprecipitant [glycogen, linear acrylamide, SeeDNA (Amersham Biosciences, Freiburg, Germany); n = 12] on the RNA recovery and the inhibition of the subsequent reverse transcription and PCR processes. In the third experiment, the collection/storage of single oocytes was compared [RNAlater� (Applied Biosystems, Darmstadt, Germany) or liquid nitrogen; n = 20]. The use of the coprecipitant linear acrylamide (100% v. 80% for glycogen and 58% for SeeDNA; P ≤ 0.05) and the storage in liquid nitrogen (100% v. 84% for RNAlater; P ≤ 0.001) showed the highest RNA recoveries without inhibition. Furthermore, we analyzed (with the now optimized protocol) single blastomeres derived from 8- (n = 6) and 16-cell (n = 6) IVF embryos by mechanical treatment. The aim of this experiment was to analyze the expression divergences in blastomeres from normal cultured embryos without synchronization and, further, how many single blastomeres per embryo are required to obtain a comparable expression level of all blastomeres. The results showed that nearly 50% of an embryo was required (at least 4 blastomeres from an 8-cell and 7 blastomeres from a 16-cell embryo). These findings are mainly caused by different cell cycle phases of the analyzed blastomeres. Further studies with synchronized blastomeres are in progress. In conclusion, the present study demonstrates an effective RNA isolation method for a reliable qPCR analysis of single blastomeres. This work was supported by grant of the Deutsche Forschungsgemeinschaft (DFG) (FOR 478/1).

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