Improm 2 reverse transcriptase system kit

Specific pathogen-free (SPF) Penaeus monodon (~4 g, fresh weight) were purchased from the Shrimp Genetic Improvement Center, BIOTEC, Thailand. Shrimp were reared in an aerated tank with seawater at 20 ppt salinity for 7 days before the experiments began. The hemolymph was collected and total RNA was extracted using the TRI Reagent (Molecular Research Center) following the manufacturer’s protocol. First-strand cDNA was synthesized using the ImProm-II Reverse Transcriptase System kit (Promega) with 1.5 μg of total RNA and 0.5 μg of oligo(dT)₁₅ primer. For the gene expression analysis at different developmental stages, RNA extraction and cDNA synthesis as described above was done on three individual shrimp from four stages including nauplius 3 (N3), protozoea 2 (Z2), mysis 2 (M2) and post-larvae 15 (PL15).

To validate the RNAseq results, quantitative real-time PCR (qPCR) were carried out to determine gene expression levels of the 10 selected genes. Gene-specific qPCR primers were designed by using Primer3 (Untergasser et al., 2012 (link)) (Table S2). Total RNA (1.5 µg) was reverse transcribed into cDNA using an ImPromII™ Reverse Transcriptase System kit (Promega, USA) according to the manufacturer’s recommendation. Each 20 µL qPCR reaction included 0.5 µL cDNA, 200 nM of each primer, and SsoAdvanced™ Universal SYBR^® Green supermix (Bio-Rad, USA) according to the company’s instruction. The thermal cycling parameters were 95 °C for 30 s, followed by 40 cycles of 95 °C for 15 s, 57 °C for 30 s and 72 °C for 30 s. The melting curve analysis was performed from 65 °C to 95 °C with a continuous fluorescent reading with a 0.5 °C increment. The threshold cycle (Ct) was analyzed using BioRad CFX Manager 2.1 software (Bio-Rad, USA).

The total RNA was extracted from various rice tissues with WelPrep total RNA isolation reagent (WELGENE, Republic of Korea), according to the manufacturer’s instructions, and treated with RNase-free DNase I (Takara Bio, Shiga, Japan) to prevent genomic DNA contamination. First-strand cDNA was synthesized from 2 µg of total RNA in a 25-µL reaction mixture with using the ImProm II Reverse Transcriptase system kit (Promega, Madison, WI), followed by quantitative real-time PCR (qRT-PCR) analysis to determine gene expression levels (Bio-Rad, CFX96 Touch Real-Time PCR Detection System, USA) using a SYBR premix ExTaq kit (Takara Bio, Shiga, Japan). The gene expressions were normalized using the rice ubiquitin gene (LOC_Os06g46770) as an internal control. Changes in expression were calculated via the ΔΔ_Ct method. Primers for PCR are listed in Supplementary Table 5.

Total RNA was isolated from leaves of Col, ein2/ore3, and ahk3/ore12 plants using WelPrep™ Total RNA Isolation Reagent (Welgene, Korea) and used for cDNA synthesis employing the ImProm II™ Reverse Transcriptase System kit (Promega, USA). Quantitative real-time PCR (qPCR) was performed using SYBR Premix Ex-Taq (Enzynomics, Korea) and the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). Fold changes in gene expression were calculated using the C_T method relative to their initial expression in leaves of each genotype at day 0 (before dark incubation) in dark-induced leaf senescence or 12 days after leaf emergence (DAE) in developmental leaf senescence and normalized against Actin-2 (ACT2). Primers used for qPCRs are listed in Supplementary Table S1 at JXB online.

Total cellular RNA was extracted from cells by use of the RNeasy mini kit (Qiagen, Alameda, CA), then reverse-transcribed to cDNA by use of the Improm-II Reverse Transcriptase system kit (Promega, Madison, WI). The PCR primers were as follows:
HPV-16E7, forward, 5′-ATGAGAGGAAACAAC CCAAC-3′
HPV-16E7, reverse, 5′-AGCTAGGGCACACA ATGGTA-3′
β-actin, forward 5′-TGGCATTGCCGACAGGATG CAGAA-3′
β-actin, reverse 5′-CTCGTCATACTCCTGCTTG CTGAT-3′
Quantitative real-time PCR of CIP2A, c-Myc and E2F1, E2F2, E2F3 levels were performed as described [40 (link)] using the following oligos:
CIP2A, forward, 5′-GCCACACTGATTCGGTG TTTT-3′
CIP2A, reverse, 5′-TGCCGACAAAGATTTGCC AATA-3′
c-Myc, forward, 5′-GTCAAGAGGCGAACACA CAAC-3′
c-Myc, reverse, 5′-TTGGACGGACAGGATGT ATGC-3′
E2F1, forward, 5′-CATCCCAGGAGGTCACT TCTG-3′
E2F1, reverse, 5′-GACAACAGCGGTTCTT GCTC-3′
E2F2, forward, 5′-ACGGCGCAACCTACAA AGAG-3′
E2F2, reverse, 5′-GTCTGCGTGTAAAGCG AAGT-3′
E2F3, forward, 5′-GGTCCTGGATCTGAACA AGGC-3′
E2F3, reverse, 5′-CCTTCCAGCACGTTG GTGAT-3′
GAPDH, forward, 5′-GCACCGTCAAGGCTGA GAAC-3′
GAPDH, reverse, 5′-TGGTGAAGACGCCAG TGGA-3′