M mlv reverse transcription

Total RNA was extracted and purified using standard methods (Life Technologies; RNA Easy, Qiagen, Valencia, CA, USA). M-MLV reverse transcription (Promega) was utilized to synthesize cDNA. 5 lncRNA expressions in sinonasal tissues were measured by qRT-PCR which was performed on the ABI 7500 qPCR system with the primer pairs listed in Table 2. The raw quantifications were normalized to the beta-actin gene values for each sample and fold changes were shown as mean ± SD in three independent experiments, each in triplicate.

The total RNA was prepared using TRIzol^® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Subsequently, the cDNA was synthesized at 42 °C for 30 min by M-MLV reverse transcription (Promega, Madison, WI, USA). PCR was performed using specific primers designed to amplify the open reading frame (ORF) of genes and probable splice form of Masc, as well as to detect the sex-specific pattern of Bmdsx under different conditions. The corresponding primers for RT-PCR are shown in Supplementary Table S1. For qPCR, the reactions were run on an ABI7500 Real-Time PCR machine (Applied Biosystems, Foster City, CA, USA) with SYBR^® Premix Ex Taq™II (TaKaRa, Kusatsu, Japan). The eukaryotic translation initiation factor 4A (silkworm microarray probe ID sw22934) was used as the internal control [18 (link)]. We used the 2^−ΔΔCt method to analyze data.

Ten pairs of hippocampal tissues were used for qRT-PCR validation. After RNA isolation, M-MLV reverse transcription (Promega, Madison, WI, USA) was used to synthesize cDNA. Quantitative PCR analysis and data collection were performed on the ABI 7500 qPCR system (Applied Biosystems, Foster City, CA, USA). Primer pairs are listed in Table 1. Relative gene expression was calculated using the 2^−ΔΔCt method, and fold changes are shown as means ± standard deviation (SD) from three independent experiments. U6 and β-actin were used as references.

QRT-PCR was performed to verify the differential expression of lncRNAs and mRNAs between atherosclerotic plaque tissues of ApoE⁻/⁻ mice fed with a HFD for 12 weeks and normal aortic tissues. Total RNA extracted for microarray analysis was reverse-transcribed to cDNA using M-MLV reverse transcription (Promega, Madison, WI, USA) according to manufacturer’s instructions. QRT-PCR was performed using an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) and a SYBR Green kit (Takara, Dalian, China) using the primer pairs listed in Table 1. We used 18S RNA as an endogenous control for normalization. For relative quantification, 2^−ΔΔCt was calculated and used as an indication of gene relative expression.Table 1

Primer sequences used for qRT-PCR

Gene symbol	Forward	Reverse
Cldn1	ATCCACAGTCCCTCGTAG	GGTTTCATCCTGGCTTCT
Ccl8	ATACCCTGCTTGGTCTGG	GCTCATAGCTGTCCCTGTC
Spp1	ACCATGCAGAGAGCGAGGATT	GGGACATCGACTGTAGGGACG
Traf3	TCCGAGGTATCCACTATGA	TGTCGCCCAAACTGTTCT
Fas	TTCGTGAAACTGATAAAAACTGC	TCTGATGGTCTCCAAAATGCT
mapk3	GGCTTTCTGACGGAGTATGTGGC	TGGTCCAGGTAGTGCTTGCCG
NONMMUT000151	GCATCAATAGTCCCAGTAA	GATGGAGGTCTGGTTTGT
NONMMUT042398	GATCACTACAGCCTCCTC	CTGGTAAATCAGGCAACT
Gm13241	TTTGAAACCTCCTCCCTA	ATGCCCAGTGGTGTATTT
NONMMUT019491	ACCCTGCTTCTATTCCAC	GTAGGCAAACACCCAGAC
NONMMUT001754	CGAAATGAAGCGTAGTGT	TTGACAACCAGGAATAGC
NONMMUT051707	AACTAGGCTCACTGAATAAA	TGTAACTGGGAAGGAAGA

Total RNA was extracted from taro leaf samples in RNAlater^® using the RNeasy Plant Mini Kit and synthesis of cDNA was performed using M-MLV reverse transcription (Promega, United States; Wang et al., 2017 (link)). A universal potyvirus-specific ELISA (Agdia Inc., USA) and a universal potyvirus-specific RT-PCR (Zheng et al., 2010 (link)) were used to test two samples with feathery mottling and distorted leaves, and one asymptomatic sample. RT-PCR with dasheen mosaic virus (DsMV) CI-specific primers and a DsMV-specific triple-antibody sandwich ELISA (Agdia Inc., United States; Wang et al., 2017 (link)) were used to confirm DsMV in taro leaf samples.
We used the CTAB method to isolate total DNA from 100 mg banana leaf samples with banana bunchy top virus (BBTV) symptoms kept in RNAlater^® using CTAB method and were tested by PCR using BBTV-Rep gene-specific primers and CP-specific primers BBTV-HAF1 and BBTV-HAR1 (Hamim et al., 2017 (link)). Six samples were also tested for BBTV using a BBTV-specific antibody in a triple-antibody sandwich-ELISA (Agdia, Elkhart, Inc.).