Mircute plus mirna first strand cdna kit

After 48 h of adipocyte treatment with bta-miR-149-5p mimics or mimics NC (negative control), the adipocytes were collected and RNA was extracted through RNAiso Plus kit (Takara Beijing, China). The RNA integrity both quantity and quality were evaluated through optical density (OD) of 260 and ratio of the OD of 260/280 with Nano Quant plate TM (TECAN, Infinite M200 PRO). The RNA quality was further analyzed through 1% agarose gel. Then, the RNA was subjected to reverse transcription for cDNA synthesis through PrimeScriptTM RT reagent kit (perfect real time) with a gDNA eraser (Takara, Beijing, China). The qRT-PCR was performed using SYBR^® Premix Ex Taq II kit (Takara, Beijing, China). For the bta-miR-149-5p expression analysis, we used a mircute miRNA isolation kit (TIANGEN, Beijing, China). The first strand cDNA library was constructed using miRcute Plus miRNA First-Strand cDNA kit (TIANGEN, Beijing, China). The bta-miR-149-5p expression was measured through mircute plus miRNA qPCR detection kit (TIANGEN, Beijing, China). For gene expression analysis, GAPDH and β-actin were used, while for miRNA expression, U6 was used as housekeeping genes. The 2^−ΔΔCt method was used for the identification of relative mRNA and miRNA expression levels [39 (link)]. The PCR primer sequences are shown in Table S1.

We randomly selected two circRNA-miRNA-mRNA axes and examined the relative expression levels of the circRNAs, miRNAs and mRNAs in these axes with respect to the conidial and mycelial stages. The primer sequences are listed in Additional file 14: Table S13. qRT-PCR of mRNA was performed with reagents and procedures identical to those described above for circRNAs. The procedure for miRNA extraction was as follows: total RNA was extracted using a miRNeasy Mini Kit (Qiagen), and gDNA was eliminated using a TURBO DNA-free™ Kit (Thermo Fisher Scientific) at 37 °C for 30 min. MiRNA was then reverse-transcribed with a miRcute Plus miRNA First-Strand cDNA kit (TIANGEN, Beijing, China) in a 20 μl reaction volume at 42 °C for 60 min and 95 °C for 3 min. qRT-PCR of miRNA was performed using miRcute SYBR Green Master Mix (TIANGEN). The reaction conditions were 15 min at 95 °C followed by 40 cycles of 20 s at 94 °C and 34 s at 60 °C. U6 was used as the internal control of miRNAs for gene expression normalization. Each experiment was performed with three replicates. The relative expression level and statistical significance were calculated as stated above.

Trizol (Invitrogen, CA, USA) was utilized for extracting total RNA in myocardial tissues. The concentration and quality of RNA were determined by NanoDrop2000 (Thermo Fisher Scientific, MA, USA). Expression of LncRNA and mRNA was quantified using PrimeScript™ RT reagent Kit (TaKaRa, Dalian, China)  and TB Green™Premix Ex Taq™ II (TaKaRa). miRNA expression was calculated using miRcute Plus miRNA First-Strand cDNA Kit and miRcute Plus miRNA qPCR Kit (SYBR Green) (Tiangen) (Fu et al. 2020 (link)). The primers were composed by BGI Co. (Shenzhen, Guangdong, China) (Additional file 3: Table S1). U6 was the endogenous control of miR-185-3p while glyceraldehyde phosphate dehydrogenase (GAPDH) was that of LINC00461 and Myd88. Data were reckoned by 2^−ΔΔCt method (Livak and Schmittgen 2001 (link)).

The total RNA was exacted using the miRcute miRNA isolation kit, quantified using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), and reverse-transcribed to cDNA using the miRcute Plus miRNA First-Strand cDNA kit (TIANGEN Biotech) for miRNA. The RT-PCR for miR-155-5p was measured using a commercial kit containing primers for miR-155-5p and U6 (TIANGEN Biotech, Beijing, China). The endogenous control used for the miRNA normalization was U6 small nuclear. The primers were found to be compliant with the MIQE guidelines, based on their Ct values determined with a standard curve.