Quantiscript reverse transcriptase kit

Following the manufacturer’s instructions, RNA was extracted with an RNeasy Mini kit obtained from Qiagen (Hilden, Germany) in an RNase-free environment. A Nano Drop 2000 spectrophotometer (Thermo Fisher Scientific, USA) was used for the determination of the amount and purity of RNA. One µg of RNA was reverse transcribed by Quantiscript reverse transcriptase Kit supplied by Qiagen. The polymerase chain reaction was completed using a Rotor Gene Q thermocycler (Qiagen) and SYBR Green PCR Master Mix (Qiagen). Quantitative PCR was performed in triplicate and included no template controls. The sequences of the PCR primer pairs used: cyclin D1, 5′-TGCTTGGGAAGTTGTGTTGG-3′ (forward) and 5′-AATGCCATCACGGTCCCTAC-3′ (reverse); GAPDH, 5′-TCAAGAAGGTGGTGAAGCAG-3′ (forward and 5′-AGGTGGAAGAATGGGAGTTG-3′ (reverse). The relative gene expression was assessed by the comparative cycle threshold (Ct) (2^−ΔΔCT) method and values were normalized to the expression of GAPDH.

To determine the response of tomato plant to carrageenan treatment following viroid inoculation, the transcript abundance of tomato defense genes TomLOX (forward primer, 5′-GGCCACGTTGACCTC CGCAA-3′; reverse primer, 5′-TGCGCTGAAGCCAGCCAGAT-3′, TomAOS (forward primer, 5′-ACACGACGCCGTTTTCGAGGTG-3′; reverse primer, 5′-CCGACGAACCGATCGGCGAC-3′ and PR1 (forward primer, 5′-GACGAGTTGGCGTTGGCCCT-3′; reverse primer, 5′-AGCGGCTAGGTTTTCGCCGT-3') were analyzed by quantitative real time PCR. Leaf tissues were sampled at 0 and 7 dpi and total RNA was extracted using a plant RNA extraction kit (Qiagen, Mississauga, ON, Canada). The quality and quantity of RNA was assessed with Nanodrop ND-1000 (NanoDrop Technologies Wilmington, DE, USA) and formaldehyde gel electrophoresis. DNAase treated RNA was used to synthesize cDNA using Quantiscript reverse transcriptase kit (Qiagen, Mississauga, ON, Canada) following manufacturer’s instructions. Real-time PCR was performed on StepOne™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR green dye with Rox (Roche Diagnostics, Mississauga, ON, Canada) according to manufacturer instructions. Transcript abundance of each selected gene was normalized to18S ribosomal RNA. Data were analyzed from three independent Real-Time PCR runs.