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Markers of Sensitivity and Resistance to EGFR Inhibitors in Colorectal Cancer
Published in Sherry X. Yang, Janet E. Dancey, Handbook of Therapeutic Biomarkers in Cancer, 2021
Jose G. Monzon, Janet E. Dancey
Real time PCR with post-PCR melting curve analysis, detects the disrupted affinity of two DNA chains caused by a mutation. A single strand of DNA containing a mutation has an altered binding affinity when bound to either a complementary mutant (MT) strand or wild-type (WT) strand, such that they dissociate at a different temperature compared with the WT double stranded sequence. Hence, real time PCR is performed using probes complementary to the WT sequence and flanking the mutation site (codons 12 and 13). Following PCR, the amplicon melting temperature is determined, defined as the temperature at which 50% of the amplicons have dissociated into single stranded DNA. Based on the different binding affinities of the WT/WT, WT/MT, and MT/MT double strands, different melting temperatures are detected. This method demonstrates a high sensitivity for the detection of KRAS mutations that was similar to direct sequencing [62]. However, if an altered melting temperature is detected (suggesting a mutant sequence), the exact nucleotide change is still not identified, and hence direct sequencing is still required.
Methods in molecular exercise physiology
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Adam P. Sharples, Daniel C. Turner, Stephen Roth, Robert A. Seaborne, Brendan Egan, Mark Viggars, Jonathan C. Jarvis, Daniel J. Owens, Jatin G. Burniston, Piotr P. Gorski, Claire E. Stewart
Melting curve analysis is also performed to distinguish a gene products ‘melting’ temperature (Tm). This so-called ‘melt curve analysis’ can be used to verify that only one intended gene target/gene of interest was amplified, indicating PCR product homogeneity (or specificity) and that the sample was not contaminated. To generate the melting curve, fluorescence of the sample product is measured as the temperature is gradually increased. A sharp increase in fluorescence is observed as the product is amplified followed by a sharp decrease in fluorescence that occurs as the product denatures, marking its ‘melting’ temperature (Figure 2.7). If there are multiple peaks, this may suggest contamination or unspecific amplification, or that primers have been unable to anneal to the target gene (Figure 2.7). Multiple smaller peaks at lower temperatures can also suggest primer-dimer issues where forward and reverse primers with complimentary sequences may have annealed to each other (or themselves) rather than the gene of interest. If this occurs, these samples cannot be used in any analyses, and primers may need to be redesigned until the melt curve analysis demonstrates primer ‘specificity’ (amplification of only one product in the melt curve analysis) following RT-qPCR. Products can also be sequenced to confirm that the gene amplified is indeed the gene of interest. Alternatively, the size of the PCR product can be confirmed by running the products on an agarose gel followed by ethidium bromide binding and UV light visualisation against known DNA molecular weight markers (see gel electrophoresis section below).
Evaluation of transcriptomic molecular classification, biological behavior, and clinicopathological features in hepatocellular carcinoma
Published in Expert Review of Molecular Diagnostics, 2023
Ruonan Chen, Zixiong Zhou, Yi Chen, Aimin Huang, Lihong Chen
The qPCR procedures were performed as previously described [13] using Bestar™ SYBR Green qPCR MasterMix kit (DBI-2043, DBI Bioscience, Shanghai, China). Briefly, total RNA was isolated from tissues using the TransZol Up Plus RNA Kit (ER501-01, TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. RNA quality was verified by ND-2000 (Nanodrop Products, Wilmington, DE, USA), with only high-quality RNA samples (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RIN ≥ 8, 28S:18S ≥ 1.0) used for analyses. The RNA was converted to complementary DNA by reverse transcriptase using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) and qPCR analysis was performed. Specificity was verified by melting curve analysis. We used 18S was used as an internal control, and gene expression analysis was performed using Applied Biosystems StepOne version 2.3 software. The primers are listed in Table 1.
Circulating microRNAs as potentially new diagnostic biomarkers of idiopathic sudden sensorineural hearing loss
Published in Acta Oto-Laryngologica, 2020
Sun Mok Ha, Kyu Rin Hwang, Il Hwan Park, Sunyoung Park, Jin Sil Choi, Dong Jun Park, Jeong-Eun Park, Su Hoon Lee, Hye Young Lee, Young Joon Seo
Real-time quantitative PCR was performed using the miRCURY LNA PCR assay (QIAGEN, Hilden, Germany) with CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The microRNAs hsa-miR-16-5p, hsa-miR-103a-3p, hsa-miR-183-5p, hsa-miR-205-5p, hsa-miR-34a-5p, hsa-miR-15a-5p, hsa-miR-23a-3p, hsa-miR-210-3p, hsa-miR-18b-5p, and hsa-miR-143-3p were used for the test. A total reaction volume of 10 µL comprising 3 µL of 1:10 diluted cDNA, 5 µL of THUNDERBIRD® SYBR® qPCR Master Mix (TOYOBO, Osaka, Japan), 1 µL of miRCURY LNA PCR assay primer, and 1 µL of nuclease-free water was used for real-time PCR. Real-time PCR was performed with pre-amplification at 95 °C for 2 min, followed by 40 cycles of a polymerase chain reaction at 95 °C for 10 s and 56 °C for 60 s. A melting curve analysis was also performed. Normalization of microRNA expression levels was performed based on two reference microRNAs, miR-103 and miR-16. The gene expression level was calculated as follows: (Expression level) = 2 × (–(Ct of the gene of interest − Ct of miR-103 or miR-16)), where Ct is the PCR cycle threshold.
Prenatal Diagnosis and Screening of Thalassemia Mutations in Bangladesh: Presence of Rare Mutations
Published in Hemoglobin, 2020
Md. Abdul Aziz, Waqar A. Khan, Bilquis Banu, Sudipta A. Das, Salma Sadiya, Sayeda Begum
DNA extraction was done on blood samples and also on fetal samples collected by CVS or amniocentesis using PureLink™ Genomic DNA Purification Mini Kit (Invitrogen, Carlsbad, CA, USA). The extraction method relies on phase separation by centrifugation of a mix of the aqueous sample. The kit was used according to the manufacturer’s instructions. The extracted DNA was tested to determine the purity and concentration of DNA per microliter volume through Qubbit® 2.0 Fluorometer (Invitrogen). The real-time polymerase chain reaction (qPCR) method was done by SYBR Green I dye for detecting some common mutations [IVS-I-5 (G>C), codon 26 (or Hb E), −90 (C>T) (HBB: c.-140C>T), codons 41/42, codon 30 and codons 8/9]. Mutation confirmation was done by melting curve analysis. Every case of PND was cross checked using the Sanger sequencing method.