Rneasy rna cleaning kit

Total RNA was extracted from these materials using TRIzol Reagent (Invitrogen, USA) following the manufacturer’s protocol. The quality of total RNA was determined using a NanoDrop Spectrometer (ND-1000 Spectrophotometer, Peqlab). The mRNAs were isolated from total RNAs using the PolyATtract mRNA Isolation Systems kit (Promega) and condensed using the RNeasy RNA cleaning kit (Qiagen, Germany); their concentration and purity were determined using the Agilent 2100 Bioanalyzer (RNA Nano Chip, Agilent). The mRNAs were fragmented and retrieved using an RNA Fragment reagent kit (Illumina) and RNeasy RNA cleaning kit (Qiagen). Then, random primers and M-MLV were used to synthesize the first chain, and DNA Polymerase I and RNase H were used to synthesize the second chain. Finally, the cDNAs were retrieved using the RNeasy RNA cleaning kit (Qiagen, Germany), and their quality was checked using the Agilent 2100 Bioanalyzer. All procedures were performed according to the manufacturers’ instructions.

Mixed larvae from the first instar to the prepupal stage, pupae, and adults (females and males) were prepared for RNA extraction. Total RNA was isolated from homogenized sample in TRIzol reagent (Takara, Dalian, Liaoning, China) following the manufacturer’s protocols. The concentration of total RNA was quantified with a Qubit3.0 (Thermo Fisher Scientific, Waltham, MA, USA) and an Agilent2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). UV absorption values at 260 nm/280 nm was recorded to monitor the purity of the RNA products (Nanodrop2000, Thermo Fisher Scientific, Waltham, MA, USA). After RNA extraction, mRNAs were purified using the interaction of the poly (A) tails and magnetic oligo (dT) beads and collected using RNeasy RNA reagent. Mixed mRNAs were fragmented into 300–800 bp pieces using RNA fragment reagent (Illumina), and the pieces were collected using an RNeasy RNA cleaning kit (Qiagen). Subsequently, RNA fragments were copied to make first-strand cDNA using MMLV reverse transcriptase (Takara, Dalian, Liaoning, China) and random primers. Second-strand cDNA synthesis was performed using DNA Polymerase I and RNase H. The Illumina HiSeq2000 system and 125 paired-end reads were used for sequencing. Statistical analysis of the sequence lengths was performed to ensure sequence purity.

Eggs, mixed larvae from the first instar to the prepupal stage, pupae, and adults (females and males) from emergence to mating (3 days old) were prepared for RNA extraction. Total RNA was extracted using TRIzol reagent (Qiagen, China Shanghai). The concentration of total RNA was quantified using a spectrophotometer (Implen, Westlake Village, CA), and the RNA integrity was tested using 1.5% agarose gel electrophoresis. After RNA extraction, mRNAs were purified using a Poly A T tract mRNA isolation system and collected using an RNeasy RNA reagent. Mixed mRNAs were fragmented into 300–800 bp pieces using RNA Fragment reagent (Illumina), and the pieces were collected using an RNeasy RNA cleaning kit (Qiagen). Subsequently, RNA fragments were copied into first strand cDNA using MMLV reverse transcriptase (TaKaRa, Dalian Liaoning, Chinese) and random primers. Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. The Illumina HiSeq2000 system and 100 paired-end reads were used for sequencing. Clean reads were obtained by removing adaptors, low-quality reads, and contaminated reads from raw sequence reads. Statistical analysis of the sequence length was performed to ensure sequence purity.