1st strand cdna synthesis kit

Cells were collected, and total cellular RNA was extracted using TRI Solution (BSK-BIO, Daegu, Korea), according to the manufacturer’s instructions. cDNA was synthesised with 1 μg of total RNA using the 1st Strand cDNA Synthesis Kit (TaKaRa Bio Inc., Shiga, Japan). Real-time qPCR was performed using StepOnePlus (Applied Biosystems, Foster City, CA, USA) with SYBR Premix Ex Taq (Takara Bio Inc., Otsu, Shiga, Japan). The threshold cycle (Ct) values obtained for each reaction were normalised using the GAPDH Ct values. The sequences of primers used for real-time qRT-PCR are outlined in Table 1. All reactions were performed in triplicate.

DSM 7029 mutant cells were harvested at an OD₆₀₀ value of approximately 4.3 ± 0.5, at approximately 18 h. RNA extraction was carried out with an RNAprep Pure Cell/Bacteria Kit (TIANGEN, China) according to the manufacturer’s instructions. Genomic DNA was removed by using DNA Eraser supplied in the 1st Strand cDNA Synthesis Kit (Takara), and its removal was confirmed by PCR. cDNA was synthesized using HiScript^R III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme Biotech, China) for RT-PCR with 800 ng of total RNA. Gene expression was analysed using real-time RT-PCR with ChamQ™ Universal SYBR^R qPCR Master Mix (Vazyme Biotech, China). PCR was conducted in a BIO-RAD Real-Time System with the following thermal cycling program: 30 s at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 60 °C. The mraW gene was used as the normalization signal. The amplification primers used for each gene are listed in Additional file 1: Table S3. The transcription levels of fdx_0135, fdr_0130, and fdr_7100 were measured.

Total RNA was extracted from different tissues (heart, brain, liver, kidney, lungs, and spleen) in adult and aging mouse hearts using the RNAprep pure Tissue Kit (#DP431; Tiangen). The first cDNA strand was synthesized using a 1st-Strand cDNA Synthesis Kit (RR036A; Takara), and then real time PCR was performed using the SYBR PrimeScript RT-PCR Kit (RR820A; Takara). All of the procedures were performed according to the manufacturer’s instructions. Primers of mouse Omi/HtrA2 and GAPDH for QRT-PCR (SYBR) were designed and synthesized as follow: GAPDH-F: 5’-CTCTCTGCTCCTCCCTGTTCC-3’; GAPDH-R, 5’-CGTTCACACCGACCTTCACC-3’; Omi-F, 5’-ATCTCCTTTGCCATCCCTTC-3’; Omi-R, 5’-GGTCAGCATCATCACTCCAA-3’. The expression of Omi/HtrA2 mRNA in different tissues was calculated by the 2^−ΔΔCt method, with GAPDH as internal control.

The Total RNA Kit II (Omega Bio-Tek, Norcross, GA, United States) was used to isolate the total RNA from larvae and pupae of L. striatellus, and first strand cDNA synthesis was performed using the 1st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan). Two pairs of intermediate fragments primers were designed from the L. striatellus transcriptome database by local blast (Table 1). The full-length complementary DNA (cDNA) of LsFoxO was cloned using the rapid amplification of cDNA ends RACE kit (Takara, Japan), and the evolutionary analyses were conducted using MEGA software, version 4.0.

Environmental DNA was extracted from all samples using Qiagen’s All Prep DNA/RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Additionally, using the same kit, total RNA was extracted from all the samples of station DY12. Three subsamples of total RNA from each layer of DY12 were reverse-transcribed into cDNA using the 1st strand cDNA synthesis kit (TAKARA BIO INC., Japan) with the random primers provided in the kit.

Total RNA was extracted from neonatal rat myocardium and siRNA-treated cells at 48 hrs using TRIzol reagent (Invitrogen) as previously described 19 (link). Reverse transcription PCR (RT-PCR) was conducted using the 1st Strand cDNA Synthesis Kit (TAKARA, Dalian, China) following procedures outlined in the manufacturer’s instructions. Real-time PCR was performed with the SYBR® Premix Ex Taq™ II kit (TAKARA) to identify gene expression. Relative expression levels of target genes were calculated by calibrating to the house-keeping gene β-actin as 2-(target gene Ct-reference gene Ct). All of the RT-PCR primer sequences are shown in Table2.

The analysis of miRNA expression was performed using poly(A)-tailed real-time RT-PCR method as described in literature [59 (link)]. Firstly, Small RNA was isolated from calli and protoplasts with RNAiso for Small RNA(TaKaRa) according to the manufacturer’s instructions. Then the Small RNA were polyadenylated by poly(A) polymerase, and recovered with phenol/chloroform extraction and ethanol precipitation. Subsequently, the RNA was reverse-transcribed into cDNA with poly(T) adapter according to PrimeScript^TM 1st strand cDNA synthesis Kit and then the qRT-PCR was performed on a BIO-RAD CFX96TM Real-Time System with the SYBR®Premix Ex TaqTM II (Tli RNaseH Plus) (TaKaRa) according to the manufacturers’s instructions. The 5.8S rRNA gene was used as the reference gene. All primer sequences were listed in S1 Table. The conditions for the PCR amplification were as follows:45 cycles of 95°C for 30 s, 95°C for 10 s, 60°C for 15 s, and 72°C for 20 s. The relative expression ratio was calculated using the ΔΔCt method.