cDNA was synthesized with 2 μg of total RNA using SuperScript II Reverse Transcriptase (Roche, Basel, Switzerland) and random hexamers primers for IGF2BP1 gene or oligodT for Myc gene according to the manufacturer’s instructions. Amplification reaction assays contained 1x SYBR Green PCR Mastermix (Applied Biosystem) and primers (IDT, Coralville, IA, USA) at the optimal concentrations. Primers for detection of mRNA were as follows IGF2BP1: 5′-CAGGAGATGGTGCAGGTGTTTATCC-3′ and 5′-GTTTGCCATAGATTCTTCCCTGAGC-3′, c-myc: 5′-CAGCTGCTTAGACGCTGGATT-3′ and 5′-GTAGAAATACGGCTGCACCGA-3′ [26 (link)]. The RNA expression level was measured using the threshold cycle method [45 (link)] using 23 kDa as the normalization reference.
Superscript 2 reverse transcriptase
Manufactured by Roche
Sourced in Switzerland
Superscript II reverse transcriptase is a lab equipment product manufactured by Roche. It is an enzyme used in the process of reverse transcription, which involves converting single-stranded RNA into complementary DNA (cDNA).
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Steponeplus rt pcr system, by Thermo Fisher Scientific (2 mentions)
Random primer, by Roche (1 mentions)
Geneamp pcr system 9700, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Express sybr green reagents, by Thermo Fisher Scientific (1 mentions)
7900 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Imagequant software, by Cytiva (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
7 protocols using superscript 2 reverse transcriptase
1
Quantifying IGF2BP1 and Myc mRNA
2
Laser-Microdissected Cell RNA Extraction
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The extraction of total RNAs from clinical samples of laser-microdissected cells was performed as described previously [8 (link)]. The amplified RNA or total RNA was reverse transcribed to generate single-stranded cDNAs using a random primer (Roche) or oligo(dT)16 primer with Superscript II reverse transcriptase (Roche). We prepared appropriate dilutions of each single-stranded cDNA for subsequent PCR amplification by monitoring tubulin, alpha 3 (TUBA3) as a quantitative control. The primer sequences were 5’-CTTGGGTCTGTAACAAAGCATTC-3’ and 5’-AAGGATTATGAGGAGGTTGGTGT-3’ for TUBA3 and 5’-GTCCTGAAAGTCAAGCACCTG-3’ and 5’-GAAGTTCTTGTTGGTGCTTATGG-3’ for C16orf74. All reactions involved initial denaturation at 94°C for 2 min, followed by 22 cycles (for TUBA3) or 28 cycles (for C16orf74) of 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min on a GeneAmp PCR system 9700 (PE Applied Biosystems).
3
RNA Extraction and qPCR Analysis of PDLSCs
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Total RNA was extracted from PDLSCs using TRIzol reagent (Invitrogen, USA). Reverse transcription was performed with 1,000 ng of total RNA and SuperScript II Reverse Transcriptase (Roche) according to the manufacturers’ instructions. Quantitative polymerase chain reaction (qPCR) was performed with SYBR Ex Taq (Roche) according to the manufacturers’ instructions. Sequences of the primers are listed in Table 1 .
4
Quantitative RT-PCR Gene Expression
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RNA was extracted using TRIzol (Invitrogen) and complementary DNA was made with random hexamers and Superscript II reverse transcriptase (Roche). Quantitative real‐time PCR was performed in duplicate with Express SYBR Green reagents (Invitrogen) on the StepOnePlus RT‐PCR system (Applied Biosystems), and data were normalized using Cyclophilin A as reference gene. Primer sequences are available upon request.
5
PBMC RNA Extraction and RT-qPCR Analysis
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For isolation of total RNA from peripheral blood mononuclear cells (PBMC), whole blood collected in an EDTA Vacutainer™ tubes were firstly treated with red blood cell lysis buffer (Legend). Remaining leukocytes were recovered by centrifugation. After washing with phosphate buffer saline (PBS), the leukocyte pellet was processed for purification of total cellular RNA with Trizol (Invitrogen). RNA was reverse‐transcribed with Superscript II reverse transcriptase (Roche) into cDNA according to manufacturer's instruction. cDNA derived from 1 µg of total RNA served as a template for real‐time PCR. The expression levels of VEGFA (Hs00900055_m1) and GAPDH (Hs03929097_g1) were quantified by real‐time PCR using Taqman (Lifescience) with a fluorescence detection monitor 7900 Real‐time PCR system (Applied Biosystems). Mean Ct values were calculated for each molecule using 2−ΔCt method, where Ct values of the molecules were normalized to that of GAPDH.
6
Quantitative RT-PCR for Gene Expression
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RNA was extracted using Trizol (Invitrogen) and complementary DNA was made with random hexamers and Superscript II reverse transcriptase (Roche). Quantitative real-time polymerase chain reaction (PCR) was performed in duplicate with Express SYBR GreenER reagents (Invitrogen) on the StepOnePlus RT-PCR system (Applied Biosystems), and data were normalized using HPRT as a reference gene. Primer sequences are available on request.
7
RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted using the RNeasy Mini kit (Qiagen, Hilden, Germany). Reverse transcription was performed from 1 μg RNA using the Superscript II Reverse Transcriptase (Roche Applied Science, Basel, Switzerland) and random primers. Real Time-PCR was carried out on cDNAs using primers listed in S2 Table and the 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Results were normalized using the GeNorm software (http://medgen.ugent.be/Bjvdesomp/genorm/ ). Statistical analysis was performed using the Mann-Whitney test. Semi-quantitative PCR were performed using PATCHED1 primers F: 5′-GATAAGAGCTCCGGGGGATTC-3′ and R: 5′-CACAGTAGCTTAGGCTTCAGCCC-3′, and GAPDH primers F: 5’-CCAAGGCTGTGGGCAAGGTCAT-3’ and R: 5’-TGACAAGGTGCGGCTCCCTAGG-3’ as described [27 (link)]. PCR products were quantified after electrophoretic separation in 2% agarose gel, and SYBR fluorescence scanning (STORM; Molecular Dynamics, Sunnyvale, CA) using the ImageQuant software (Amersham Biosciences, Amersham, United Kingdom).
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