Maxwell® 16 LEV simplyRNA Purification Kit

Protocol tips

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- This kit can be used for the isolation of RNA from 5 million cells

Upstream tips
- This kit can be used for the isolation of RNA from 5 million cells

Publication protocol

Upon the completion of incubation, the culture supernatant was removed and cells were washed three times in sterile phosphate-buffered saline (PBS) to remove any conidia that did not bind/attach to cells. Total RNA was prepared from infected and uninfected A549 cells with TRIzol (Sigma Chemical Co., St. Louis, MO) in the dish (1ml TRIzol per 10 cm2), respectively. The samples were stored at -80°C prior to RNA extraction. RNA extraction and RNA-Seq analysis were performed by BGI Tech company (Shenzhen, China). Briefly, after the total RNA extraction and DNase I treatment, the mRNA was fragmented into short fragments. Then cDNA was synthesized using the mRNA fragments as templates. After agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used in quantification and qualification of the sample library. At last, the library could be sequenced using Illumina HiSeq 2000.

Primary sequencing data that produced by Illumina HiSeq 2000, is called as raw reads. Before doing any further analysis, quality control was required in order to detect whether the data is qualified. In addition, filtering of raw data was needed to decrease data noise. Several softwares updated were used to perform these tasks. After filtering, the remaining reads were called "clean reads" and used for downstream bioinformatics analysis. Clean reads were mapped to a reference sequences using SOAPaligner/SOAP2. No more than 5 mismatches were allowed in the alignment. The gene expression level was calculated by using RPKM method (Reads per kilobase transcriptome per million mapped reads), and p value was calculated according to the Poisson distribution. We used p value < 0.05 and the fold change ≥ 1.5 as the threshold to judge the significance of gene expression difference. Further, we used DAVID online tool (http://david.abcc.ncifcrf.gov) to perform Gene Ontology (GO) enrichment analysis and KEGG Pathway enrichment analysis [27–28]. Our dataset were deposited in SRA repository, and the accession number is SRP051309.

Total RNA was isolated from 1×106 A549 cells by RNApure Tissue Kit (with DNase I) (CW BIOtech, CW0560, China) following the manufacturer’s protocols. RNA was used to synthesize cDNA using Transcriptor First Strand cDNA Synthesis Kit (Roche, Cat.No.04896866001) according to the manufacturer’s protocols. For RT-PCR, A549 cells were cultured on 35-mm cell-culture plates for 24 h in Dulbecco’s modified Eagle’s medium (DMEM); 1.2 ml of Buffer RL (CW BIOtech, CW0560, China) was added to each plate, and RNA was isolated according to the protocol supplied by the manufacturer. To ensure the quality of the isolated RNA, the RNA samples were analyzed by gel electrophoresis. Total RNA (2μg in a total volume of 20μl) was denatured at 65°C for 10 min and used for reverse transcription as described by the standard protocol (Roche). cDNA (2μl in a total volume of 20μl) was used as the template for qPCR. Glyceraldehyde phosphate dehydrogenase (GAPDH) was the reference gene. These genes specific primers were designed using Primer5 software (Table 1). And the specificity of the primers were tested by NCBI Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). qRT-PCR was performed using SYBR Premix Ex Taq (Tli RNaseH Plus) (TaKaRa, cat.#RR420A, Japan) according to the manufacturer’s protocols and reaction was performed by Bio-Rad iQ5 Detection System (version:2.0.148.60623). The thermal cycling conditions were: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 10 s and 55°C for 30 s, then followed by 95°C for 1 min and 55°C for 1 min. We used the “delta delta Ct” method for analyzing the qRT-PCR data. The fold change was derived by calculating the ratio between the experimental group and control.

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