Both mRNA and miRNA were isolated using a total RNA isolation kit (Norgen). To determine expression levels of miR-181a, miR-181b, and miR-16 100 ng of total RNA was converted to cDNA using a Taqman Reverse Transcription Kit and Taqman miRNA specific primers (ABI). The miRNA cDNA was then PCR amplified using a Roche Lightcycler II real-time PCR machine along with miRNA specific Taqman probes and ABI universal UNG master mix. To determine mRNA expression levels 1000 ng of total RNA was reverse transcribed into cDNA using the Transcriptor Universal cDNA Synthesis Kit (Roche). cDNA was then PCR amplified using a Roche Lightcycler II real-time PCR machine along with gene specific PCR primers and LightCycler® 480 SYBR Green I Master Mix (Roche). Primers used are listed in Supplementary Table 1 .
Lightcycler 2 real time pcr machine
Manufactured by Roche
The Roche LightCycler II is a real-time PCR machine designed for quantitative analysis of nucleic acid samples. It enables real-time monitoring of amplification of DNA or RNA targets during the PCR process. The LightCycler II utilizes a thermocycler and integrated optics system to detect fluorescent signals generated during the amplification reaction.
Automatically generated - may contain errors
2 protocols using lightcycler 2 real time pcr machine
1
Quantifying miRNA and mRNA Levels
2
Quantitative Analysis of miRNA and mRNA Expression
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Both mRNA and miRNA were isolated using a total RNA isolation kit (Norgen). To determine expression levels of miR-181a, miR-181b, and miR-16 100 ng of total RNA was converted to cDNA using a Taqman Reverse Transcription Kit and Taqman miRNA specific primers (ABI). The miRNA cDNA was then PCR amplified using a Roche Lightcycler II real time PCR machine along with miRNA specific Taqman probes and ABI universal UNG master mix. To determine mRNA expression levels 1000 ng of total RNA was reverse transcribed into cDNA using the Transcriptor Universal cDNA Synthesis Kit (Roche). cDNA was then PCR amplified using a Roche Lightcycler II real time PCR machine along with gene specific PCR primers and LightCycler® 480 SYBR Green I Master Mix (Roche). Primers used are listed below.
List of primer sequences used in qtRT-PCR analysis.
| 18S RP | GGAAAGCAGACATTGACCTCAC |
| 18S LP | CCATCCTTTACATCCTTCTGTCTGT |
| CEBPA RP | TTCACATTGCACAAGGCACT |
| CEBPA LP | ACGATCAGTCCATCCCAGAG |
| PPARG RP | CCATTACGGAGAGATCCACG |
| PPARG LP | AGGCCATTTTGTCAAACGAG |
| ADPN RP | GAACACCTGTGAGGTCACCC |
| ADPN LP | CTGTACCCTGCCTGTGGAAT |
| PLIN RP | AGGTCTTCTGGAAGCATTCG |
| PLIN LP | CAGTCAACAAAGGCCTCACC |
| GLUT4 RP | CCCCAATGTTGTACCCAAAC |
| GLUT4 LP | CTTCCAACAGATAGGCTCCG |
| FABP4 RP | TGATGATCATGTTAGGTTTGGC |
| FABP4 LP | TGGAAACTTGTCTCCAGTGAA |
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