Multiscribe reverse transcriptase from the high capacity cdna reverse transcription kit

Total RNA was isolated from the floral tissue using Qiagen RNeasy Plant kits following the manufacturer’s instruction (QIAGEN, Valencia, CA, USA). Genomic DNA contamination was removed by on-column DNase I digestion (Qiagen Inc., Valencia, CA, USA). The concentration and RNA purity was assessed based on an absorbance ratio of 1.8 to 2.0 at 260/280 nm and ≥ 2.0 at 260/230 using the Biotek Synergy H1 Hybrid Multi-Mode Reader (BioTek Instruments Inc., Winooski, VT, USA). DNA-free total RNA (1 µg) was used for the cDNA synthesis using MultiScribe™ Reverse Transcriptase from the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, USA) in a 20 µL reaction following the manufacturer’s instruction. The MultiScribe™ reaction mix includes random primers to make cDNAs. The final cDNA products were diluted 20-fold before use in real-time PCR.

Assessment of the effect of the NM_001349884.2:c.517+5C>A mutation in DRAM2 was performed as follows. Three milliliters of peripheral blood were collected from patients and a healthy control in Tempus Blood RNA tubes and total leukocyte RNA was extracted with the Tempus Spin RNA Isolation kit (Applied Biosystems), according to manufacturer's instructions. Two micrograms of RNA were used as a template for cDNA synthesis, by the use of random primers and the MultiScribe Reverse Transcriptase from the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCRs were obtained by using primers 5′-AACAACCCTTTTTGCTGCAC-3′ (CR-7950) and 5′-GGGGTTCCAATGGAGTTTCT-3′ (CR-7951) lying on DRAM2 exons 7 and 8, respectively, according to standard cycling conditions. Resulting PCR products were resolved on agarose gels and sequenced by the Sanger technique, following their extraction and purification by the GenElute Gel Extraction Kit (Sigma-Aldrich).