M mulv cdna synthesis kit

The isolated RNA was reverse transcribed into cDNA by an M-MuLV cDNA Synthesis Kit (Sangon Biotech). The primers of this study for qRT-PCR were designed by Primer Premier 5.0 software to verify the relative expression level of randomly selected genes with the 18S gene as an internal control. The relative expression of these randomly selected genes was calculated with the 2^−△△Ct method. The PCR conditions were set as follows: predenaturation at 95°C for 10 min and 40 cycles of 95°C for 10 s and 60°C for 30 s (Wu et al., 2021 (link)).

From goat SHF bulges and stem cells, the total RNA was isolated with the RNAiso reagent kit (TaKaRa, Dalian, China). Based on the use of random primers, the first strand cDNA was reversely transcribed by M-MuLV cDNA Synthesis Kit (Sangon, Shanghai, China). One Step PrimeScript microRNA cDNA synthesis kit (TaKaRa, Dalian, China) was used to transcribe the cDNA for the microRNAs testing. We carried out real-time PCR amplification using SYBR Green I assay (TaKaRa, Dalian, China). All primers were designed through the use of Premier Primer 5.0 program (Premier Biosoft International, Palo Alto, CA, USA). The analyzed miRNA mature sequences were obtained from the miRNA database (http://www.mirbase.org, accessed on 28 July 2019), and their sense primers were designed based on the the corresponding miRNA sequence. Whereas, the corresponding anti-sense primers were provided in the kits (TaKaRa, Dalian, China), which are universal reverse primers for all miRNAs analyses. Here, all primers are listed in Supplementary Table S1 with their detailed information. The PCR reaction of each sample was performed in triplicate.

Here, RNAiso kits (TaKaRa, Dalian, China) were utilized for extracting
the total RNA from SHF bulges of cashmere goats and its stem cells. For the
expression detection of circRNA-0100 and related gene mRNAs, random primers
were utilized to synthesize the first strand cDNA with an M-MuLV cDNA synthesis
kit (Sangon, Shanghai, China), whereas for the expression detection of
miRNAs, the first strand cDNA was synthesized by a One-Step PrimeScript
microRNA cDNA synthesis kit (TaKaRa, Dalian, China). The divergent primers
for detecting the expression of circRNA-0100 were designed using the
CircPrimer program (Zhong et al., 2018). The convergent primers for mRNA
detection were designed by the Premier Primer 5.0 program (Premier Biosoft
International, Palo Alto, CA, USA). A combined internal control consisting
of UBC, YWHAZ, and SDHA was utilized for normalizing the gene expression
level (Bai et al., 2014). The corresponding mature sequences of all detected
miRNAs were retrieved from the miRNAsong database
(https://www2.med.muni.cz/histology/miRNAsong/index.php, last access: 16 April 2021). A combined
internal control consisting of let-7d-5p, miR-26a-5p, and miR-15a-5p was
utilized for normalizing the miRNA expression level (Bai et al., 2016). All
primers are provided in Table 1 with the corresponding detailed
information.