5 mm glass beads (BioSpec) for 10 min. The lysates were then incubated for 10 min at room temperature, after
which LY2835219 datasheet 150 μl of chloroform was added per ml of Tri-Reagent used. The mixtures were then centrifuged for 5 min at 4000 × g. For each sample, the aqueous phase was recovered and transferred to a clean RNase-free test tube. After two consecutive extraction cycles with acidic phenol:chloroform (1:1) and centrifugation at 4°C for 5 min, the RNA was precipitated by adding two volumes of isopropanol and incubating at room temperature for 10 min. Once precipitated, the RNA was washed with 75% ethanol, suspended in RNase-free H2O and quantified by determination of the absorbance at 260 nm in a double beam Shimadzu UV-150-20 spectrophotometer. The synthesis of cDNA was performed using 5 μg of total RNA, 1.25 μM oligo-dT18 primer, 0.5 μM dNTPs and 200 units of M-MLV reverse transcriptase (Invitrogen) in a final volume of 20 μl, according to the enzyme manufacturer’s recommended protocol. Quantitative RT-PCR Relative mRNA expression levels were determined in a Mx3000P quantitative PCR system (Stratagene) using 1 μl of the reverse transcription reaction, 0.25 μM of each primer and 10 μl of the
Copanlisib SensiMix SYBR Green I (Quantace) kit in a final volume of 20 μl. The primers used to determine the relative levels of expression are detailed in Table 1. All of the primer pairs used to amplify each gene had efficiencies greater than 95%, Thiamine-diphosphate kinase as determined by standard curves, with correlation coefficients (R2) ≥ 0.996. Table 1 Primers used in this work Primer Gene Direction Sequence (5′ to 3′) Location mactF-RT act F CCGCCCTCGTGATTGATAAC Spanning exons 2 & 3 mactR-RT act R TCACCAACGTAGGAGTCCTT Spanning exons 4 & 5 mmcrtYBF2-RT crtYB(mm) F TCGCATATTACCAGATCCATCTGA Spanning exons 1 & 2 mmcrtYBR2-RT crtYB(mm) R GGATATGTCCATGCGCCATT Exon 2 amcrtYBF-RT crtYB(am) F GTGTGCATATGTGTTGCAACCA Spanning exon 1 & intron 2 amcrtYBR-RT crtYB(am) R AGAAGGTGCCTAGTTGCCAAGA Exon 3 mmcrtIF-RT crtI(mm)
F CATCGTGGGATGTGGTATCG Spanning exons 1 & 2 mmcrtIR-RT crtI(mm) R GGCCCCTGATCGAATCGATAA Spanning exons 3, 4, 5 amcrtIF-RT crtI(am) F CGTGGTTTAATCCGTATCAGC Spanning exon 1 & intron 1 amcrtIR2-RT crtI(am) R TCTCGAACACCGTGACCT Exon 2 mcrtSF-RT crtS F ATGGCTCTTGCAGGGTTTGA Spanning exons 6 & 7 mcrtSR-RT crtS R TGCTCCATAAGCTCGATCCCAA Spanning exons 8 & 9 grg2real FW1 grg2 F CATCAAGACCTCTGTCACCAAC Spanning exons 1 & 2 grg2real RV1 grg2 R TTGGCGTCAGACGAGGACT Exon 3 BIBW2992 concentration pdcreal FW1 PDC F TCAACACTGAGCTGCCCACT Spanning exons 5 & 6 pdcreal RV1 PDC R ATTCCGAATCGGGAAGCACA Exon 6 F: Forward, R: Reverse; (mm): mature transcript, (am): alternatively spliced transcript. The Ct values obtained for each reaction were normalized to the respective value for the β-actin gene and were later expressed as functions of the control conditions using the ΔΔCt algorithm .