7500 qpcr

For RNA extraction, 1- to 4-d-old male adults were entrained to a 12:12 h LD cycle for 7 d at 29°C and then collected on dry ice at ZT 6. Total RNA was extracted from 30 heads of adult males of the Control (repo>LacZ), Glioma (repo>UAS-dEGFR^λ, UAS-dp110^CAAX), and repo>UAS-dEGFR^λ, UAS-dp110^CAAX, elav-LexA, lexAop-Rheb genotypes after 7 d of glioma development. RNA was extracted with TRIzol and phenol chloroform. Total RNA concentration was measured by using NanoDrop ND-1000. cDNA was synthetized from 1 mg of total RNA using M-MLV RT (Invitrogen). cDNA samples from 1:5 dilutions were used for real-time PCR reactions. Transcription levels were determined in a 14-ml volume in duplicate using SYBR Green (Applied Biosystem) and 7500 qPCR (Thermo Fisher Scientific). We analyzed transcription levels of ImpL2, dRheb, and Rp49 as housekeeping gene reference.
Sequences of primers were RP49 F: GCATACAGGCCCAAGATCGT, Rp49 R: AACCGATGTTGGGCATCAGA, ImpL2 F: CCGAGATCACCTGGTTGAAT, ImpL2 R: AGGTATCGGCGGTATCCTTT, dRheb F:CGACGTAATGGGCAAGAAAT, and dRheb R: CAAGACAACCGCTCTTCTCC.
After completing each real-time PCR run, outlier data were analyzed using 7500 software (Applied Biosystems). Ct values of duplicates from three biological samples were analyzed calculating 2DDCt and comparing the results using a t test with GraphPad (GraphPad Software).

The mRNA for all samples was extracted from adult brains and processed in parallel. For this, 1- to 4-day-old male adult mice were maintained at 29 °C for 7 days and collected on dry ice at ZT6. Total RNA was extracted by triplicate from 30 heads. RNA was extracted with TRIzol and phenol chloroform. cDNA was synthetized from 1 µg of RNA and cDNA samples from 1:5 dilutions were used for real-time PCR reactions. Transcription levels were determined in a 14 µL volume in duplicate using SYBR Green (Applied Biosystems, Waltham, MA, USA) and 7500 qPCR (Thermo Fisher Scientific, Waltham, MA, USA). We analyzed transcription levels of cry using Rp49 as a housekeeping gene reference.
Sequences of primers were as follows.
After completing each real-time PCR run, with cycling conditions of 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 55 °C for 1 min, and outlier data were analyzed using 7500 software (Applied Biosystems, Waltham, MA, USA). Ct values by triplicate of duplicates from three biological samples were analyzed calculating 2DDCt.