Invitrogen cdna synthesis kit

For this study 20 seeds for each of the cultivar Hong Ke Zi (cold tolerant) and BTx623 (cold sensitive) were planted in ragdoll set up under cold stress temperatures. The temperatures were continuous 14 °C for cold stress using Conviron walk-in growth cabinets. The seedlings were allowed to grow under 14 h light/10 h dark conditions. Leaf tissue samples for RNA extraction were obtained from five seedlings per replicate for Hong Ke Zi and BTx623. Three replicates were extracted separately using the TRIzol reagent and company recommended protocols (Life Technologies, Grand Island, NY). The total RNA samples from each replicate were quantified using Nano Drop and subsequently purified using the RNAeasy mini clean up kit (Qiagen, Valencia, CA). To evaluate the expression differences observed, qRT-PCR primers were designed for four selected genes in the expression module using primer3 (Additional file 7) and sorghum actin gene was used for normalization. Briefly, qRT-PCR was performed using copy-DNA libraries generated from the total RNA using Invitrogen cDNA synthesis kit (Invitrogen, Grand Island, NY). qRT-PCR was performed on the cDNA of the four samples with 3 biological and 3 technical replicates using SybrGreen on LightCycler 480.

Total RNA was isolated using the Qiagen RNeasy mini kit (Qiagen, Toronto, ON). To quantify gene expression levels, equal amounts of cDNA were synthesized using the InVitrogen cDNA synthesis kit (InVitrogen, Carlsbad, CA) and mixed with the hot start reaction SYBR Green I PCR master mix (Roche Diagnostics, Indianapolis, IN, USA) containing 10 μM of the primers (BioCorp; Dollard-Des Ormeaux, Quebec) described in Supplementary Table 2. Psmb6 or RPLP was amplified as an internal control. All qPCR reactions for mouse primers were conducted using the LightCycler^® 480 Instrument (Roche Life Science, Indianapolis, IN, USA) at 95°C for 5 min, followed by 45 cycles of 95°C for 10 s, 60°C for 10 s and 72°C for 10 s. Using primers for the human cDNAs, the reactions were performed at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s, 60°C for 20 s and 72°C for 20 s. The specificity of the reaction was verified by melting curve analysis. The relative quantification of each mRNA was performed using the comparative Ct method. Data processing was performed using LightCycler^® 480 software (Roche Life Science).