Experion rna stdsens analysis kit

Approximately 200–300 mg of the muscle powder was pulverized in the presence of liquid nitrogen using a mortar and pestle. The total RNA was extracted using Trizol^® reagent (Invitrogen, San Diego, CA, USA) followed by isolation using PureLink^TM Micro-to-Midi RNA isolation system kit (Invitrogen, San Diego, CA, USA) following instructions of the manufacturer. The yield and integrity of the extracted RNA were determined using Experion^TM automated electrophoresis system (BIORAD, Gladesville, NSW, Australia) and Experion^TM RNA StdSens analysis kit (BIORAD, Gladesville, NSW, Australia). Samples displaying RNA quality indicator (RQI) ≥ 8.0 and 28S:18S ribosomal RNA ratio close to 1.5 were considered sufficient quality RNA. One microgram total RNA from each sample was reverse transcribed into cDNA using the SuperScript^® III First-Strand Synthesis System (Invitrogen, San Diego, CA, USA) for RT-PCR and 50 ng/μL random hexamer primer (Invitrogen, San Diego, CA, USA), according to manufacturer’s instructions into a final volume of 20 μL and stored at −80 °C.

The P. pastoris GS115 microarray encompasses well-known and predictable P. pastoris GS115 genes and transcripts. Coupled with NCBI gene prediction processes and Agilent's probe selection, the design delivers increased data quality as well as less redundant gene coverage. The 5040 P. pastoris GS115 genes and transcripts were annotated. Each gene has one probe and most probes have 3 replications. The sequence content was sourced from NCBI BioProject PRJNA39439. All the representative probes were designed by Agilent's eArray. The sequence orientation, accuracy, and clustering assembly classification was validated with P pastoris GS115.
Cells were subjected to further extractions, and 9 mL of culture were mixed with 5 mL of freshly prepared chilled 5% (v/v) phenol (Sigma) solution in absolute ethanol, centrifuged at 4°C and 12, 000 rpm for 5 min. The harvested cells were stored at -80°C until extraction.
The RNA extractions were performed with RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol of enzymatic extraction using lyticase (Sigma). RNA samples were quantified and analysed for purity using Experion RNA StdSens Analysis Kit (Bio-Rad) with a RQI between 8.8 and 9.9. The GenomeOligo microarray of P pastoris was custome designed by Agilent corporation, and the detailed description is provided in S1 File.

For RNA extraction, the TRIzol reagent (Ambion) was used in combination with the PureLink RNA minikit (Ambion). Briefly, cells were disrupted in TRIzol containing 0.2 g of 0.2-mm-diameter glass beads (acid washed; Sigma) using a FastPrepH-24 instrument (MP Biomedicals), and the following extraction steps were performed according to the manufacturer’s instructions. DNase treatment was performed according to the on-column PureLink DNase treatment protocol (Life Technologies/Invitrogen). RNA was quantified on a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc.), and the quality and integrity were checked using the Experion RNA StdSens analysis kit (Bio-Rad).
The transcriptomes of the Synechocystis wild-type and kpsM mutant strains were analyzed by RNA sequencing (RNA-seq), using three biological replicates. RNA-seq data were generated by Novogene. A total amount of 3 to 5 μg RNA per sample was used as the input material for the RNA sample preparations. A detailed description of the procedure can be found in Text S1. The distribution of the identified proteins into functional categories was performed as described above.

Fat body tissues, excluding gonads, were collected in triplicates immediately after dissection in ice chilled TRIzol® Reagent (Invitrogen, cat. no. 10296-028). After collecting fat body from 40 individuals for each replicate, samples were vortexed and stored at −80°C. After thawing the total RNA was prepared according to the instructor's manual for TRIzol® Reagent and further purified with the help of the Qiagen RNeasy Mini Kit (cat. no. 74104). Qiagen DNase for DNA degradation and subsequent purification was applied. cDNA was prepared employing the SuperScript III Reverse Transcriptase (Invitrogen, cat. no. 18080-044). Quality was assessed using the Experion RNA StdSens Analysis kit (BioRad).