Total RNAs were isolated using Trizol (Invitrogen Life Technologies), according to the manufacturer’s instructions. One microgram total RNA was used for reverse transcription, using a ReverTra Ace qPCR-RT Kit (Toyobo, Osaka, Japan) in a reaction volume of 10 μL. qRT-PCR was performed to detect the relative level of XIAP mRNA, using the Light-Cycler rapid thermal cycler system 2.0 (Roche Diagnostics Ltd, Burgess Hill, United Kingdom) with SYBR Green Master Mix (Toyobo). Primers used for real-time PCR are as follows: XIAP (forward) 5′-CCGTGCGGTGCTTTAGTTGT-3′ (reverse) 5′-TTCCTCGGGTATATGGTGTCTGAT-3′; GAPDH (forward) 5′-TGGTCACCAGGGCTGCTT-3′ (reverse) 5′-AGCTTCCCGTTCTCAGCCTT-3′. Real-time qRT-PCR was performed with cycle parameters of 95°C for 5 minutes, followed by 40 cycles of 95°C for 15 seconds, 58°C for 20 seconds, and 72°C for 20 seconds. The relative concentrations of the PCR products were calculated using LightCycler System software. All samples were normalized to GAPDH expression, using the 2−ΔΔCt or 2−ΔCt method.20 (link),21 (link) All experiments were conducted in triplicate.
Light cycler rapid thermal cycler system 2
Manufactured by Roche
Sourced in United Kingdom
The Light-Cycler rapid thermal cycler system 2.0 is a laboratory instrument used for real-time polymerase chain reaction (PCR) analysis. It is designed to rapidly perform thermal cycling for the amplification and detection of DNA or RNA samples.
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Lab products found in correlation
Sybr green master mix, by Toyobo (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Revertra ace qpcr rt kit, by Toyobo (1 mentions)
Spectramax microplate spectrophotometer, by Molecular Devices (1 mentions)
Taqman mirna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Mirna specific taqman mirna assay kit, by Thermo Fisher Scientific (1 mentions)
7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
2 protocols using light cycler rapid thermal cycler system 2
1
Quantitative Analysis of XIAP mRNA Expression
2
Quantification of miR-7 and Bcl-2 mRNA
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Total RNA isolation from tissues or cells was performed using mirVana miRNA Isolation Kit (Applied Biosystems/Ambion, Austin, TX, USA) according to the manufacturer's protocol. RNA concentrations were measured using the SPECTR Amax microplate spectrophotometer (Molecular Devices Corp). Total miRNA from tissues was extracted by using the mirVana miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's instructions. cDNA was synthesized from 5 ng of total RNA by using the Taqman miRNA reverse transcription kit (Applied Biosystems, Foster City, CA), and the expression levels of miR-7 were quantified by using miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems). Additionally, qRT-PCR was performed to detect the relative level of Bcl-2 mRNA, using the Light-Cycler rapid thermal cycler system 2.0 (Roche Diagnostics Ltd, Burgess Hill, United Kingdom) with SYBR Green Master Mix (Toyobo). qRT-PCR was performed by using the Applied Biosystems 7500 Sequence Detection system. The expression of miR-7 was defined based on the threshold cycle (Ct), and relative expression levels were calculated as 2 -((Ct of miR-7)-(Ct of RNU6B)) after normalization with reference to expression of RNU6B small nuclear RNA. The expression of Bcl-2 mRNA was normalized to GAPDH expression, using the 2 -ΔΔCt or 2 -ΔCt method as previously described [12] .
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