To minimize the least direct effect of treatment, the T24R2 resistant cell line was used to extract RNA and protein after leaving at least two weeks interval from the removal of the treating drug from T24R2. Total RNA was extracted from T24R2 cells using the RNeasy Mini kit (Qiagen, Valencia, CA, USA) and the same way with those from T24 cells. cDNA was produced from 1 µg of total RNA using the oligo(dT) primer and Omniscript reverse transcriptase enzyme (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. qRT-PCR was done with the FastStart Universal SYBR Green Master (Roche Diagnostics, Indianapolis, IN, USA), a ready-to-use hot start reaction mix using a 7500 real-time PCR system (7500 realtime PCR system, Applied Biosystems, Foster City, CA, USA). GAPDH was used as the reference gene, and foldchange in gene expression was calculated making use of the comparative CT (2-ΔΔCT) method. Primer sequences are presented in Fig. 1 .
Omniscript reverse transcriptase enzyme
Manufactured by Qiagen
Sourced in United States
Omniscript Reverse Transcriptase is an enzyme used in the reverse transcription process, which converts RNA into complementary DNA (cDNA). This enzyme is essential for various molecular biology applications that require the analysis of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using omniscript reverse transcriptase enzyme
1
Quantifying Drug Resistance Gene Expression
2
Measuring Muscle Transcripts Abundance
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The muscle biopsies specimens frozen immediately in liquid nitrogen were used for measuring abundance levels of selected transcripts. Frozen tissue samples were homogenized in 1 mL Trizol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted according to the manufacturer protocol. RNA concentration was measured using a ND-1000 Spectrometer (NanoDrop Technologies Inc., Montchanin, DE, USA). Reverse transcription was performed with Omniscript Reverse Transcriptase enzyme (Qiagen Inc., Valencia, CA, USA) at 37°C for 60 minutes. The qPCR reactions were performed using Assay-On-Demand TaqMan probes (VEGFA, Hs00900055_m1; FLT1, Hs01052961_m1; KDR, Hs 00911700_m1; HIF1A, Hs00153153_m1; MFN1, Hs00966851_m1; MFN2, Hs00208382_m1, OPA1, Hs01047018_m1) according to the manufacturer's protocol (Applied Biosystems, Foster City, CA, USA) and were run on the CFX96 Real-Time system (Bio-Rad, Foster City, CA, USA). Expression of the hypoxanthine-guanine phosphoribosyltransferase (HPRT1, Hs01003270_g1) transcript with stable levels following experimental conditions was quantified to control for variation in cDNA amounts. The abundance of RNA was calculated as 2-(threshold cycle). Data were normalized to the expression levels of the HPRT1 mRNA.
3
Quantitative RNA Expression Analysis
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Total RNA was extracted from the serum of control and patient samples, and then cDNA synthesis was performed using the Omniscript Reverse Transcriptase enzyme produced by Qiagen, Germany, and on the template strand. The concentration and quality of the extracted RNA were measured by reading the absorption intensity (OD) using a spectrophotometer (Nanodrop). The quantitative PCR method or Real-time PCR based on TaqMan primers and probes is a particular method to count the number of copies of RNA or DNA in a sample (12) (Table 1).
TaqMan probes are hydrolytic probes designed to increase the specificity of quantitative PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as a reference gene, and by taking into account the average CT values related to the target genes and their difference, we obtained the ΔΔCT level.
TaqMan probes are hydrolytic probes designed to increase the specificity of quantitative PCR. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was used as a reference gene, and by taking into account the average CT values related to the target genes and their difference, we obtained the ΔΔCT level.
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