Minibest rna extraction kit

RNA was extracted from frozen mouse hippocampus using MiniBEST RNA Extraction Kit and protocols (TaKaRa, Beijing, China). An aliquot of 2-μg RNA was applied for cDNA preparation based on the standard procedures using random hexamers and PrimeScript RT Reagent Kit (TaKaRa). Expression levels of mRNAs and the housekeeper transcript β-actin were gauged by SYBR-based qRT-PCR with specific primers (Tsingke, Beijing, China). The cycle threshold (Ct) value was recorded, and relative expression was determined by the 2^-ΔΔCt method. Primer details are shown in Table 1.Table 1

Sequences of the primers used for qRT-PCR

Name (mouse)		Primers for PCR (5′-3′)
PER2	Forward	AACAAATCCACCGGCTACTG
	Reverse	CTCCGGTGAGACTCCTCTTG
PER3	Forward	AGAAGCTCCAGAGCATGGAA
	Reverse	TCTGTCTTCACAGGCGACAC
TIMELESS	Forward	CTTGCATGCAGAATGGAGAA
	Reverse	GCTCTCACCGAGGTTTTCAG
FBXL3	Forward	TGGCGATGTTTTGAATTTGA
	Reverse	TTTGATCAGCTCTGGGTGTG
NFIL3	Forward	CCATGGGTCCACTAGCAACT
	Reverse	GTTCGTCTTCCCCATCAGAA
β-Actin	Forward	CGATATCGCTGCGCTGGTC
	Reverse	AGGTGTGGTGCCAGATCTTC

A Takara minibest RNA extraction kit (Takara, Japan) was used to extract RNA from the kidneys. RNA was reverse-transcribed using a Primescript RT master mix kit (Takara, Japan). The cDNA samples were amplified in duplicate. Quantitative real-time PCR (Q-PCR) was carried out on the Gentier 96E (Tianlong Technology, Xi’an, China) with TB Green Premix Ex Taq. Q-PCR was run under the following conditions: 95 °C, 30 s; 95 °C, 5 s; 60 °C, 30 s. The primer sequences of genes such as URAT1, GLUT9, OAT1, OCT1, ABCG2, TLR4, MYD88, NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC) and Caspase-1 are listed at supporting information in Supplementary Table S1. Gene expression was normalized with β-actin, and 2^−ΔΔCt was used to calculate the result.

Total RNAs from tissues and cells mentioned above were purified by MiniBEST^® RNA Extraction Kit (TaKaRa, Japan) in an RNAse-free environment and manner. The quality of the total RNAs was detected by Nanodrop. miRNA-complementary cDNA was obtained from total RNAs by means of a PrimeScript™ RT kit (TaKaRa, Japan) according to stem-loop RT primers (RiboBio, Guangzhou, China). Then, Bio-Rad IQ5 real-time PCR was coused with SYBR Green PCR Master Mix (Bio-Rad, USA) to analyze cDNA. The PCR program was 95°C warm-up for 32 s, followed by 42 cycles of (95°C for 5 seconds, plus 61°C for 10 s). The relative gene expression was analyzed by 2^−ΔΔCt methods that normalized to the internal control GAPDH or U6. The primer sequences were shown as follows: hsa_circ_0077837 forward, 5′-CCTGGAGAAACATGCCAAGGG-3′, and reverse, 5′-TCACTTCAGACACAGAGCCTACT-3'; GAPDH forward, 5′-CGGAGTCAACGGATTTGGTC-3′, and reverse, 5′-TTCCCGTTCTCAGCCTTGAC-3'; miR-1178-3p forward, TGCTCACTGTTCTTCCC, and reverse, 5′-GAACATGTCTGCGTATCTC-3'; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′, and reverse, 5′-AACGCTTCACGAATTTGCGT-3'; APITD1 forward, 5′-GATTTTGTAAGATATATTTGAGGTAT-3′, and reverse, 5′-AACCCCCTACTCAACTTACTCTAC-3'.

MX2 mycelium grown in LM for seven days was harvested. Total RNA was isolated using MiniBest RNA Extraction kit (TaKaRa, Japan), and PrimeScript II cDNA Synthesis kit (TaKaRa, Japan) was used for synthesizing first strand cDNA according to the manufacturer’s instructions.
Based on laccase nucleotide sequence (Lac1) from T. hirsuta MX2 (GenBank accession number: MN327569), two primers of Lac1-F (5′-CGGAATTCGCCATTGGACCGAAGGCGAACCTCG-3′) and Lac1-R (5′-ATTTGCGGCCGCTCACAGATCGCCCTCCGCCAGCTTG-3′) were designed to amplify gene sequence encoding mature peptide of Lac1. Two restriction sites of EcoR I and Not I were introduced at the 5′ and 3′ ends of the PCR products, respectively. The PCR procedure was: initial denaturation at 98 °C for 10 s; 33 cycles of 98 °C for 10 s, 65 °C for 5 s, and 72 °C for 1.5 min; final extension at 72 °C for 10 min. The PCR products were analyzed on 1.5% agarose gel. The band of approximately 1.5 kb in size was purified, ligated into pMD 19-T and verified by sequencing.
To expression in P. pastoris, the sequence of Lac1 was separated from recombinant pMD 19-T vector by EcoR I and Not I restriction enzymes, and was ligated into EcoR I-Not I-digested pPIC9K, designating pPIC9K-Lac1. The pPIC9K-Lac1 was then linearized with Sac I, and was transferred into P. pastoris GS115 by electroporation. Positive transformants were selected after 3 days in MD medium.