Tb green kit

To measure the amount of total RNA, we used the TRIzol reagent (Invitrogen) to extract RNA from the various cells using NanoDrop 2000 (Thermo Fisher Scientific). The PrimeScript™ RT reagent Kit (Takara) was used to reverse transcribe 1 µg of total RNA into cDNA. qRT-PCR assays were carried out by the use of the TB Green Kit (Takara) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) as follows: denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 5 sec, 60°C for 40 sec, and 72°C for 45 sec. SHP2 primers were 5′-GGAGGAGAACGGTTTGATTCTT-3′ and 5′-CGAGTCGTGTTAAGGGGCTG-3′. β-Actin primers were 5′-CCTGGCACCCAGCACAAT-3′ and 5′-GGGCCGGACTCGTCATAC-3′, which was used as an internal control for standardization. The relative expression of genes was calculated and normalized using the delta-delta CT (2-ΔΔCt) method relative to β-actin. Independent experiments were done in triplicate.

Total RNA was extracted from different tissues (midgut, fat body, haemocytes, integument, Malpighian tubules, head and silk gland) and developmental stages (egg to adult) using Trizol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The concentration and integrity of RNA were observed at an absorbance ratio of A_260/280 and A_260/230 using a NanoDrop 2000 spectrophotometer (Thermo Scientific^TM, Waltham, MA, USA), and by 1.0% agarose gel electrophoresis, respectively. Total RNA samples were used to prepare first strand cDNA using PrimeScript^TM RT Master Mix (Takara, Dalian, China) following the manufacturer’s instructions. The qRT-PCR was performed with a TB Green kit (Takara) and analyzed with the Multicolour Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA), as previously reported [36 (link)]. The thermal cycling profile was set as follows: initial denaturation at 95 °C for 30 s and 40 cycles of 95 °C for 5 s, 60 °C for 30 s, and 72 °C for 20 s. The transcriptional level of Bmserpin2 was normalized to B. mori GAPDH. The relative expression levels were calculated using the 2^−∆∆Ct method according to a previous protocol [37 (link)]. The experiment was conducted in triplicate.

RNAs were extracted from GC cells via Trizol (Takara, Shiga, Japan); then, 1 µg RNA of each sample was applied to prepare cDNA. Subsequently, TB Green kit (RR430A; Takara) was adopted to implement qRT-PCR. Glyceraldehyde-phosphate dehydrogenase (GAPDH) and RNU6 (U6) were used as housekeeping genes. The primers were listed in Table 1. The relative expression levels were computed by the 2^−ΔΔCt method [24 (link)].Table 1.

Primer sequences used for qRT-PCR

Name		Primers (5’-3’)
circDLST	Forward	CAAAACCCCAGCGTTTGCAG
	Reverse	CACTGTTGTTAATGCTTTCTCCCA
miR-489-3p	Forward	GTATGAGTGACATCACATATAG
	Reverse	CAGTGCGTGTCGTGGAGT
EIF4A1	Forward	TCATCAACACCCGGAGGAAG
	Reverse	TGCACATCAATGCCTCTGGC
GAPDH	Forward	TCCCATCACCATCTTCCAGG
	Reverse	GATGACCCTTTTGGCTCCC
U6	Forward	CTCGCTTCGGCAGCACATATACT
	Reverse	ACGCTTCACGAATTTGCGTGTC

Total RNA from the murine liver was extracted using the Trizol reagent (TaKaRa, Dalian, China). RNA concentrations were determined by nanodrop and then normalized. cDNA was synthesized from 1 µg of total RNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa). qPCR reactions were performed using TB Green kit (TaKaRa) on an ABI 7500 real-time PCR system (Thermo Fisher, Quantstudio 3). Please see Supplementary Table S2 for a complete list of primers used in this study.

TRIzol (Invitrogen) was used to isolate total RNA from tissues or cells. Then, RNA was reverse transcribed into cDNA using the PrimeScript Reagent Kit (Takara, Shiga, Japan) according to the manufacturer's instruction. RT-qPCR was performed using the TB Green Kit (Takara) on an ABI 7500 Real-time PCR system (Applied Biosystems, Foster City, CA, USA). GAPDH was used as an endogenous control. Relative ARAF mRNA level was determined by the 2^-ΔΔCt method [15 (link)]. The primer sequences are listed as follows: ARAF forward 5′-CCCACATTCCAAGTCACCAGCA-3′ and reverse 5′-CCTCCCAGTAATAGCCTGAGTC-3′ and GAPDH forward 5′-GTCTCCTCTGACTTCAACAGCG-3′ and reverse 5′-ACCACCCTGTTGCTGTAGCCAA-3′.

Total RNA was extracted using TRIzol (Invitrogen) following the manufacturer’s instructions. The cDNAs were synthesized using First-Strand Synthesis Kit (TAKARA, Tokyo, Japan). The primers of circRNA, miRNA and mRNA were synthesized by GenePharma (Suzhou, China). Quantitative real-time PCR (qRT-PCR) was performed using the TB Green™ Kit (TAKARA, Tokyo, Japan). The expression of circRNA and mRNA were normalized relative to GAPDH, and miRNAs was normalized relative to U6, which was calculated using 2^−△△Ct method. The primers used in this study are listed in Table 1.Table 1

Primers used in this study.

Gene name	Forward (5′–3′)	Reverse (5′–3′)
hsa_circ_0069117	TGCGCATTTCAAGAAGAACA	TCCACTTCAGAGCCTCCTGT
miR-875-3p	CGCGCGCCTGGAAACACTGAG	ATCCAGTGCAGGGTCCGAGG
U6	CGCTTCGGCAGCACATATAC	TTCACGAATTTGCGTGTCATC
PF4V1	GCCAGGAGATGCTGTTCTTG	GGGAGGTGGTCTTCACACAC
AKT	AGCGACGTGGCTATTGTGAAG	GCCATCATTCTTGAGGAGGAAGT
ERK1	TACACCAACCTCTCGTACATCG	CATGTCTGAAGCGCAGTAAGATT
GAPDH	GCACCGTCAAGGCTGAGAAC	TGGTGAAGACGCCAGTGGA