Total RNA was isolated using miRNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and DNA was eliminated (Rnase-Free Dnase Set Qiagen). Reverse transcription was performed using the Omniscript RT Kit (Qiagen), TBP gene was used as reference gene for normalization using 50ng of total RNA and specific primers (EZH2: 5′-CCTGTCGACATGTTTTGGTC-3′; TBP: 5′-GTGTTTAAAATCTACATA-3′). PCR was performed in a 7500 Fast Real Time PCR System using Go Taq PCR master mix (Promega) and 1 μl of cDNA as a template. Melting curves were performed to verify specificity and absence of primer dimers. Reaction efficiency was calculated for each primer combination. The sequences of the specific oligonucleotides used have been reported elsewhere [7 (link), 8 (link)]. To measure quantitatively the expression of miRNAs, RNA was extracted using the same method as for the genes. Reverse transcription was carried out from 10 ng total RNA along with miR-specific primer using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). PCR assays were performed using TaqMan® Gene Expression Master Mix and 7500 Fast Real Time PCR System (Applied Biosystems) as reported [38 (link)]. For normalization, we used RNU6B.
7500 fast real time pcr system
Manufactured by Promega
Sourced in United States
The 7500 Fast Real Time PCR System is a thermal cycler designed for real-time PCR analysis. It can perform rapid thermal cycling and real-time detection of nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Gotaq pcr master mix, by Promega (2 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (2 mentions)
Mirneasy mini kit, by Qiagen (2 mentions)
Omniscript rt kit, by Qiagen (2 mentions)
Rnase free dnase set, by Qiagen (2 mentions)
Rnalater, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Gotaq qpcr master mix, by Promega (1 mentions)
Sybr green pcr master mix, by Promega (1 mentions)
4 protocols using 7500 fast real time pcr system
1
Quantitative Analysis of Gene and miRNA Expression
2
Quantifying TARM1 Gene Expression in Salmonella-Infected Mouse Tissues
3
Telomere Length Measurement in PBMCs
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Telomere length in PBMCs was measured using the methods described previously (Cawthon, 2009), with some modifications. We used 36B4 for reference as a single copy gene. The primer sequences (5′–3′) were: telomere forward: GGT TTT TGA GGG TGA GGG TGA GGG TGA GGG TGA GGG T; telomere reverse: TCC CGA CTA TCC CTA TCC CTA TCC CTA TCC CTA TCC CTA; 36B4 forward/reverse CAG CAA GTG GGA AGG TGT AAT CC; 36B4 forward/reverse CCC ATT CTA TCA TCA ACG GGT ACA A. The 36B4 reaction included 7.5 μL of SyBr Green PCR MasterMix (Promega, UK), 0.45 μL of primer forward, 0.75 μL of primer reverse and 3.3 μL of miliQ water. For telomere reaction: 7.5 μL of SyBr Green PCR MasterMix (Promega), 0.45 μL of primer forward, 0.45 μL of primer reverse and 3 μL of miliQ water.
The real-time polymerase chain reaction (PCR) was run with 7500 Fast Real Time PCR System (Promega, CA, USA) at 50°C for 2 minutes, then 40 cycles of 95°C for 2 minutes, 55°C for 15 seconds and 60°C for 1 minute.
The real-time polymerase chain reaction (PCR) was run with 7500 Fast Real Time PCR System (Promega, CA, USA) at 50°C for 2 minutes, then 40 cycles of 95°C for 2 minutes, 55°C for 15 seconds and 60°C for 1 minute.
4
Quantifying mRNA and miRNA Expression
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Total RNA was isolated using miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions and DNA was eliminated (Rnase-Free Dnase Set Qiagen). Reverse transcription was performed for 10 ng total RNA using the Omniscript RT Kit (Qiagen) and specific primers for each gene. The sequences of the oligonucleotides used are listed in Supplementary Table 6 . PCR was performed in a 7500 Fast Real Time PCR System using Go Taq PCR master mix (Promega) and 1 μl of cDNA template. Melting curves were performed to verify specificity and absence of primer dimers. Reaction efficiency was calculated for each primer combination, and TBP gene was used as reference gene for normalization [65 (link)]. To measure miRNA expression quantitatively, RNA was extracted using the same method as for the genes. Reverse transcription was carried out from 10 ng total RNA along with miR-specific primer using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems). PCR assays were performed using TaqMan® Gene Expression Master Mix and 7500 Fast Real Time PCR System (Applied Biosystems). For normalization, we used RNU6B. Discrimination between samples showing increased or decreased tumor/normal relative expression was calculated using the median.
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