Superscript retrotranscriptase

The pool of cDNA was synthesized by reverse-transcription of 2 °g of total RNA using SuperScript retrotranscriptase (Invitrogen) following the manufacturer’s conditions. Real time PCR was performed using the ViiA™ 7 Real-Time PCR System (Applied Biosystems). Each reaction was set up by mixing 12.5 °l of SYBR Green PCR Master Mix with 0.4 μl of 25 °M primer mixture and 10 ng of cDNA template in a final volume of 30 °l. Amplification was performed at 60°C. Negative controls with no cDNA were included. The sequences of specific primers of selected genes are defined in S1 Table. Transcript levels of target genes were normalized to those of the housekeeping gene rnpB measured with the same samples [9 (link)]. Relative quantification was performed according to the comparative Ct method (ΔΔCt Method) [10 (link)]. The minimum fold-change threshold was set up to ± 1.5 fold.

Reverse transcription of 2 μg of total RNA from each sample was carried out using SuperScript retrotranscriptase (Invitrogen) in 40 μl of reaction volume containing 150 ng of random primers (Invitrogen), 1 mM dNTP mix (GE Healthcare) and 10 mM DTT. Real-time PCR (qPCR) was performed using the QuantStudio™ 5 Real-Time PCR System (Applied Biosystems). Each reaction was set up in a final volume of 30 μl containing 10 ng of cDNA template, 12.5 μl of SYBR Green PCR Master Mix and 160 nM of each primer. Negative controls with no cDNA were included. The sequences of primers used for transcript quantification of selected genes are defined in Supplementary Table S1. Relative quantification was performed according to the ΔΔCt method (Livak and Schmittgen, 2001 (link)). Expression levels were normalized using 16S rRNA as housekeeping gene.

The effects of lindane on gene expression were analyzed in three independent cultures of Anabaena sp. PCC 7120 with an initial OD_750nm of 0.3. RNA was extracted from 25 mL of each culture after 12 and 24 h of exposure to 7 mg/L of lindane following a method described in Sarasa‐Buisan et al. (2022 (link)). The absence of DNA in the RNA samples was checked by real‐time PCR, using oligonucleotides for the housekeeping gene rnpB (Vioque, 1992 (link)). RNA was quantified spectrophotometrically using a SPECORD® PLUS Analytik Jena spectrophotometer.
Two micrograms of total RNA was reverse‐transcribed using SuperScript retrotranscriptase (Invitrogen) following the manufacturer's conditions. Real‐time PCR was performed using the ViiA™ 7 real‐time PCR System (Applied Biosystems). The specific primers for each gene that were analyzed are included in Appendix: Table A1. Each reaction was set up by mixing 12.5 µL of SYBR Green PCR Master Mix with 0.4 μL of 25 µM primer mixture and 10 ng of cDNA template in a final volume of 30 µL. The extension of PCR products was performed at 60°C. The relative mRNA levels of the target genes were normalized to the housekeeping gene rnpB (Vioque, 1992 (link)). Relative quantification was performed according to the comparative Ct method (ΔΔCt Method) (Livak & Schmittgen, 2001 (link)). The minimum fold‐change threshold was set up to ±1.5‐fold.