Quick rna miniprep kit

Cells were grown in phenol red-free DMEM supplemented with 5% charcoal-stripped (CS) fetal bovine serum (5% CS-DMEM) for 24 h. To determine baseline gene expression, cells were cultured in charcoal-stripped medium for at least 24 h to remove factors that may affect basal signaling. Cells were collected, and total RNA was extracted using the Quick RNA Mini Prep Kit in accordance with the manufacturer's protocol (Zymo Research, Irvine, CA). The quality and concentration of RNA were determined spectrophotometrically by absorbance at 260 and 280 nm using the NanoDrop ND-1000. Total RNA (1 μg) was reverse-transcribed using the iScript kit (BioRad, Hercules, CA) and qPCR was performed using SYBR-green (Bio-Rad Laboratories, Hercules, CA). Cycle numbers of ERK5-ko cells were normalized to β-actin and parental control cells scaled to 1, n = 3. For patient-derived xenografts, RNA was isolated from tumor pieces using QIAzol Lysis Reagent (Qiagen, Valencia, CA) and Quick RNA Mini Prep Kit (Zymo Research, Irvine, CA).

The quantification of the mRNA levels of the housekeeping genes GAPDH and β-ACTIN, pluripotency genes OCT4 and NANOG, GC markers FRAGILIS, PIWIL2 and STELLA, SSC markers UCHL1 and CD90 and male GC markers DAZL and STRA8, was determined using Q-PCR (Table 1). Total RNA was isolated from cells using a Quick-RNA MiniPrep kit (Zymo Research) following the manufacturer’s instructions. Total RNA was quantified using a Qubit 3.0 (Invitrogen, Fluorometer, CA, USA). Genomic DNA digestion was performed using DNase I from the Quick-RNA MiniPrep kit (Zymo Research) following the manufacturer’s instructions. The cDNA was synthesized and amplified using an Affinity Script Q-PCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA, USA), using a Step One thermocycler (Applied Biosystems, Foster City, CA, USA). The PCR reaction was performed using a Brilliant SYBR Green QPCR Master Mix kit (Agilent Technologies) and an Eco Real-Time PCR System thermocycler (Illumina, San Diego, CA, USA). Each reaction tube consists of 5 μL Sybr Green, 1 μL forward primer, 1 μL reverse primer, 2 μL nuclease-free H₂O and 5 ng cDNA. The cDNA amplification was extended for 40 cycles, and relative expression analysis was performed using the ΔΔCt (Ct: threshold value) method normalized with both GAPDH and β-ACTIN housekeeping genes [30 (link)].

For studies with BIN67, SCCOHT-1, and COV434 cells, RNA from frozen cell pellets was extracted using the Zymo Quick-RNA Miniprep Kit (Zymo cat# R1054). Total RNA was prepared using the Illumina TruSeq mRNA v2 kit with an input of 500 ng of RNA for each sample to produce unstranded RNA libraries following the manufacturer’s protocol. Final RNA libraries were quantified using the Qubit High Sense Reagent kit and Agilent Tapestation HSD1000 tapes. Libraries were equimolar pooled and paired end sequenced across three lanes of a HiSeq4000 (paired end x 78 bp).
For RNA-seq analysis of the BIN67 pIND20-A-FOS and A427 pIND20-BRG1 cell lines, RNA was extracted from frozen cell pellets in biological duplicate (BIN67 pIND20-A-FOS) and biological triplicate (A427 pIND20-BRG1) using Zymo Quick-RNA Miniprep Kit. Library preparations and RNA sequencing was performed by Novogene Inc.

We tested for the presence of poecivirus in tissue samples from the 8 AKD-affected BCCH subjected to metagenomic deep sequencing, from 11 additional BCCH with AKD, and from 9 control BCCH. Half of a mandible from each black-capped chickadee was ground using a mortar and pestle on liquid nitrogen, and RNA was extracted using a Zymo Quick-RNA miniprep kit. In some cases, the cloaca was also tested; the cloaca was homogenized using a pellet pestle, and RNA was extracted using a Zymo Quick-RNA miniprep kit and reverse transcribed as described above. Samples were screened via PCR using poecivirus-specific primers (see Table S1 in the supplemental material), and amplicons were Sanger sequenced as described above, with a slight difference in the thermocycling: the thermocycling consisted of 98°C for 30 s, 40 to 45 cycles of 98°C for 10 s, 58°C for 10 s, and 72°C for 30 s, and then a 5-min elongation step of 72°C. Positive PCR results were confirmed by Sanger sequencing. We tested two NOCR and two RBNH with elongated beaks for the presence of poecivirus in the same manner, using primers BCCHpic_1F and BCCHpic_1R (see Table S1) and BCCHpic_2F and BCCHpic_2FR.

Synthetic pre-miRNA for in vitro binding assay was designed based on miR-2022 backbone, with the mature and star stands changed to target an mCherry synthetic gene, which is not found in the Nematostella genome. To generate a control sequence, the original sequence was shuffled and non-hairpin secondary structure was validated with RNAfold web server (Lorenz et al., 2011 (link)). The reverse complement DNA templates were ordered from IDT (USA) and transcribed using the AmpliScribe T7-Flash Transcription kit protocol (Lucigen). The DNase-treated products were cleaned with Quick-RNA MiniPrep Kit (Zymo Research), then validated on agarose gel and concentration was measured with Epoch Microplate Spectrophotometer (BioTek Instruments, Inc, USA). Next, 790 ng of the products were biotinylated with Pierce RNA 3' End Biotinylation Kit (Thermo Fisher Scientific). After ligation of 15 hr, products were cleaned according to the manufacturer’s protocol and pellets were dissolved with 12.5 µl nuclease-free water. A second cleaning step was conducted with Quick-RNA MiniPrep Kit (Zymo Research). Products were eluted with 16 µl nuclease-free water and concentration was measured with NanoDrop (Thermo Fisher Scientific).