Superscript 3 reverse transcriptase

RNA was isolated from neonatal heart tissues of both groups using the TRIZOL reagent (Sigma-Aldrich, St. Louis, MO, United States) following the manufacturer’s protocol. Quantification of RNA was done with the NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, United States). DNase treatment was done before cDNA synthesis using 1 μg RNA with the SuperScript III Reverse Transcriptase (Takara Bio, San Jose, CA, United States). A reverse transcriptase-polymerase chain reaction (PCR) was carried out using the EmeraldAmp PCR Master Mix and using the following primers for target genes: Forward primer, 5′-GGAAGATCATGGAGCAGTCG-3′ and Reverse primer, 5′-GTCGGGATAATCAGCCATGT-3′ for Sox11 and Forward primer, 5′-CCACCGAGCTATCCACTCAT-3′ and Reverse primer, 5′-GTCCGGTTTCAGCATGTTTT-3′ for Mmp9. RNA expression levels were normalized using ribosomal protein L32 (Rpl32) as a reference gene using the following primer pair: Forward primer, 5′-AGATTCAAGGGCCAGATCCT-3′ and Reverse primer, 5′-CGATGGCTTTTCGGTTCTTA-3′.

Total RNA was extracted using TRIzol (15596018, Invitrogen) and converted to cDNA using Superscript III reverse transcriptase (2680, TaKaRa, Japan). The cDNA was amplified by PCR using the Premix Ex Taq™ (RR901, TaKaRa). Quantitative real-time PCR (qRT-PCR) was performed using the iQ™ SYBR^® Green Supermix Kit (170-8880, Bio-Rad, USA) on a CFX96 Touch™ real-time PCR instrument (Bio-Rad). The primer sets used in the RT-PCR and qRT-PCR are presented in Table S2.

Total RNA from bone tissues or cells was extracted with the TRIzol Reagent (Invitrogen, California, USA) according to the manufacturer’s instructions. First-strand cDNA was synthesized by incubating 1 μg of total RNA with Superscript III reverse transcriptase (Takara, Tokyo, Japan) for 1 h at 42.0 °C following oligo (dT) priming. qRT-PCR was performed using the CFX96 (BIO-RAD, California, USA) instrument and individual PCRs were performed in 96-well optical reaction plates with SYBR Green-I (Takara, Tokyo, Japan) according to the manufacturer’s instructions. Target gene expression was normalized to that of the reference gene GAPDH. The 2^−ΔΔCt method was used to calculate the relative gene expression. These PCR products were subjected to melting curve analysis and a standard curve to confirm the correct amplification. All PCRs were performed in triplicate, and the primers used for PCR are listed in Supplementary Table 1.

After culturing Schwann cells on various submicron-grooved films for 5 days, real-time quantitative polymerase chain reaction (RT-qPCR) was used to assess the expression levels of target genes. Total RNA was extracted from Schwann cells using TRIzol reagent (Invitrogen, Carlsbad, NM, USA), and 1 μg of total RNA was reverse transcribed to cDNA using Superscript III reverse transcriptase with random hexamer primers (RR037A, Takara, Beijing, China). RT-qPCR was performed on a real-time fluorescence quantitative PCR instrument (LightCycler 96, Roche, Basel, Switzerland) using the following amplification parameters: 95 °C for 2 min, 40 cycles at 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. The primers of target genes (Table 1) were synthesized by Guangzhou IGE Biotechnology Ltd., and GAPDH was used as the housekeeping gene for normalization. The fold changes in the expression of each target gene were compared by calculating 2^−ΔΔCt.

Total RNA was extracted from the fat body of each instar P. xylostella after 24h treatment with M. anisopliae using Trizol reagent according to the manufacturer’s protocol (Invitrogen, USA). First-strand cDNA was synthesized with 2μg of total RNA in combination with oligo-dT₁₈ primer and Super-script III reverse transcriptase (TaKaRa, Japan) was used to remove the genomic DNA. Px-cec2 and Px-cec3 Unigenes sequences were obtained from P. xylostella transcriptome and the PCR reactions were performed according to the following conditions: 5 min at 94°C, 30 cycles at 94°C for 30 sec, 55°C (Px-cec2) or 56°C (Px-cec3) for 30 sec, 72°C for 30 sec and 8 min at 72°C. 2 μg of mRNA was used to prepare the 5′- and 3′-RACE cDNAs using the SMART RACE cDNA Amplification Kit (TaKaRa, Japan). 3′—UTR region was amplified by 3′-RACE using the following touchdown PCR: 5 cycles of 94°C 30 sec, 72°C 3 min; 5 cycles of 94°C 30 sec, 70°C 30 sec, 72°C 1 min; 25 cycles of 94°C for 30 sec, 62°C (Px-cec2) or 65°C (Px-cec3) for 30 sec, and 72°C for 1 min, while for 5′—UTR, annealing temperature was changed to 65°C (Px-cec2) or 63°C (Px-cec3). All primers Px-cec2-F1/Px-cec-R and Px-cec3-F1/ Px-cec-R for 3′ -UTR and Px-cec-F/Px-cec-R2, Px-cec-F/Px-cec-R2 for 5′ -UTR are shown in Table 1.

Total RNA was extracted using TRIZOL™ reagent (Invitrogen, Carlsbad, CA, USA) and was reverse transcribed with (dT)n primers and Superscript III Reverse transcriptase (TaKaRa Biotechnology, Dalian, China). The following primer sequences were used:

Id1a sense: 5′-TCAACGGCGAGATCAGC-3′,

anti-sense: 5′-TCGGTCTTGTTCTCCCTCA-3′;

Id1b sense: 5′-TGCCTAAGGAGCCTGGAA-3′,

anti-sense: 5′-CCGCCTGTGAAAACGAGA-3′.

Total RNA was isolated from 3T3-L1 adipocytes and epididymal adipose tissue from mice using Trizol reagent (TAKARA, Ohtsu, Japan). Reverse transcription was performed using SuperScript III Reverse transcriptase and Oligo (dT) primer (TAKARA, Ohtsu, Japan). PCR amplification was performed with SYBR Premix Ex TaII (TAKARA, Ohtsu, Japan) using the Applied Biosystems 7500 Real-Time PCR System. Fold changes in expression were analyzed using the 2^−ΔΔ Ct relative quantitative method. This Primers used in the study were included in Supplementary Tabel S1.