Rna quantitect reverse transcription kit

Whole retinas were harvested as described above and immediately placed in RNAlater (Qiagen, Hilden, Germany; Cat# 76104). The RNeasy Mini Kit (Qiagen; Cat# 74104) was used to extract total RNA following the manufacturer’s protocol, and the quantity and quality of this RNA were estimated using a Nanodrop 1000 (Thermo Fisher). One microgram of total RNA was used as input for cDNA synthesis using the RNA QuantiTect Reverse transcription kit (Qiagen; Cat# 205311). 10 ng of cDNA was used as a template for each qRT-PCR reaction using the PowerTrack SYBR Green supermix (Applied Biosystems, Waltham, MA, USA; Cat# A46109). The Ct values for Actb were used to determine relative transcript expression levels using the 2^ΔΔCt method. Supplementary Table 1 contains a list of all primers and associated genes assayed. Got1 primers used in the rod PR-specific cKO retina were located on exons 6 (forward) and 7 (reverse).

Four biological replicates of leaf tissue were sampled from leaves of each accession at the 1^st and 5^th leaf stages. Total RNA was extracted using TRIzol® Reagent (Life Technologies) following the manufacturer’s protocol. cDNA was synthesised from 3 ug total RNA using a RNA QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). Quantitative real-time PCR (qPCR) was performed on a C1000TM Thermal Cycler (Bio-Rad, Hercules, CA, USA) with SYBR green detection (QuantiTect SYBR Green PCR Kit) with three technical replicates. Primers were developed to target mlo-11 aberrant transcripts only by amplifying the mlo-11 repeat region read-through promoter sequence with the primers MloPro_F3 and MloPro_R1. These amplify a 138 bp section upstream of the Mlo 5′ UTR. A second set of primers amplified total Mlo (Mlo and mlo-11 read-through) transcripts from an intron-spanning section of Mlo exons 6 and 7, as the mlo-11 repeat units are composed of Mlo exons one to five15 (link). These transcripts were detected with the primers MloEx6_F and MloEx7_R, which amplify a 100 bp region. Internal reference primers used were Actin F and R, as described for CNV above. Relative transcript levels were calculated by the ∆∆Ct method factoring in primer efficiencies.