For RNA analysis, iSLK.219 cells were seeded at a density of 33,000 cells per well in a 24-well plate, and the lytic cycle was induced by the addition of doxycycline (1 μg/mL). RNA samples were collected at the indicated time points in RNA lysis buffer (Zymo Research). BC3 cells were seeded at 500,000 cells per mL and induced using TPA for 48 h. One milliliter of the cells was collected, and the cell pellet was lysed in RNA lysis buffer (Zymo Research). Total RNA was extracted using the Quick-RNA MiniPrep kit (Zymo Research) by following the manufacturer’s protocol. For mRNA measurements, cDNA was prepared using an iScript cDNA synthesis kit (Bio-Rad) per the manufacturer’s protocol. In all cases, 18S rRNA levels were used as an internal standard to calculate relative mRNA levels. Real-time quantitative PCR (RT-qPCR) was performed using iTaq Universal SYBR green Supermix (Bio-Rad) in a CFX Connect real-time PCR detection system (Bio-Rad). No-template and no-RT controls were included in each replicate. CFX Manager software was used to analyze the data. Primers are listed in Table S3 .
Rna lysis buffer
Manufactured by Zymo Research
Sourced in United States
The RNA lysis buffer is a solution designed to facilitate the extraction and purification of RNA from biological samples. It is formulated to effectively lyse cells and disrupt the cellular structure, releasing the RNA for subsequent purification steps. The buffer contains chaotropic agents and detergents that help to denature proteins and disrupt cell membranes, ensuring the efficient release of RNA. This product is intended for use in RNA isolation and preparation protocols.
Automatically generated - may contain errors
Lab products found in correlation
Quick rna miniprep kit, by Zymo Research (9 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (6 mentions)
Quick rna microprep kit, by Zymo Research (5 mentions)
Quick rna microprep isolation kit, by Zymo Research (3 mentions)
Qscript cdna supermix, by Quantabio (2 mentions)
Applied biosystems powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000 sequencing system, by Illumina (2 mentions)
Dna rna shield, by Zymo Research (2 mentions)
Quick rna miniprep, by Zymo Research (2 mentions)
Itaq universal sybr green supermix, by Bio-Rad (2 mentions)
Cfx connect real time pcr detection system, by Bio-Rad (2 mentions)
Anti mouse cd16 cd32, by Thermo Fisher Scientific (2 mentions)
Anti mouse cd45 antibody clone 30 f11, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Kapa mrna hyperprep kit, by Roche (2 mentions)
Pen strep, by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Primer sequence, by Merck Group (1 mentions)
Dnaase 1, by Thermo Fisher Scientific (1 mentions)
46 protocols using rna lysis buffer
1
RNA Analysis of Viral Induction
2
RNA Isolation from Cells and Tissues
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Cells were harvested by trypsinization, washed with PBS and mixed in RNA lysis buffer (Zymo Research). Cardiac tissue was lysed in 600 µL of RNA lysis buffer (Zymo) and homogenized using TissueLyserII (Qiagen). Homogenate was spun down for 2 min at 15000 g and the supernatant was processed according to the manufacturer’s instructions. Zymo Quick-RNA MicroPrep isolation kit was used to extract total RNA. 100% ethanol was added to the lysates and samples were than processed on the columns. DNA contamination was removed by DNase I digestion. RNA was washed and eluted in 20 µl of water. RNA concentration and purity were assessed with Nanodrop 1000 (Thermo Fisher Scientific).
3
Glyco-siRNA Transfection and qPCR Analysis
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Example 14
293T cells were plated 24 hours before the experiment at 100,000 cells in 1 mL of growth media in 24-well plates. 2 μl of lipofectamine was added to 100 μl of serum free media, and glyco-siRNA duplex was added separately to serum free media to 50 nM final concentration. These two mixtures, lipofectamine and diluted duplex, were added together at room temperature and incubated for 20 minutes. Media was aspirated from plated 293 T cells and replaced with 100 μl of fresh media. 20 μl of the lipofectamine-glyco-siRNA mixture was added to each well and incubated overnight. RNA was purified from cells using RNA lysis buffer (Zymo), followed by RNA prep, wash, and elution buffers and spins at 10,000 g for 2 min each (Zymo). After RNA was quantified it was diluted to 10 ng/μL to have 50 ng per qPCR reaction. Samples were run in duplicate and each sample had a biological replicate. qPCR primers were to the beta catenin gene and PPIB and amplified using primers at 125 nM, 1×Taq polymerase master mix, MMLV RT enzyme (0.5 units per reaction), and SYBR green at 1×. Samples were first incubated for 30 min at 50° C. then 10 min at 95° C. followed by 40 cycles of 30 s of 95° C. and 1 min at 60° C. Beta catenin Ct values were normalized by those of Ct values of PPIB to report relative abundance (% beta catenin mRNA) in
4
Harvesting GFP+ Neurons for RNA Analysis
5
Siglec Expression Quantification by qPCR
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FACS-sorted cells were resuspended in RNA lysis buffer (Zymo Research, Irvine, CA) and stored at − 80 °C until RNA isolations were performed using the ZR RNA isolation kit (Zymo Research), according to the manufacturer’s instructions. The RNA was treated with DNAase I (amplification grade; Invitrogen) to remove any genomic DNA before being reverse-transcribed into cDNA, as described elsewhere [40 ]. To check for genomic DNA contamination control samples without reverse transcriptase were included. cDNA was stored at − 20 °C until further use. Real-time PCR was performed on a CFX96 system (Bio-Rad, Veenendaal, Netherlands) using SYBR Green reaction mix (Sigma-Aldrich) and Siglec expression was calculated relative to GAPDH expression. Primer sequences (Sigma-Aldrich) were derived from the Harvard Primer Bank database (Supplementary Table 1) [41 (link)].
6
RNA Extraction and Sequencing Protocol
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Following media removal, cells were washed with 1 ml of DPBS and detached by adding 400ul of 0.25% trypsin per well. Following a 10 minutes incubation at 37 °C, trypsin was neutralized with 1.6 ml of culturing media. Cell suspensions were transferred into 1.7 ml tubes and pelleted by centrifugation at 300×g for 5 minutes. Cells were then lysed in 300ul of RNA lysis buffer (Zymo Research #R1060-1-50), and stored at − 80 °C until RNA extraction was performed using the Quick-RNA MiniPrep kit (Zymo Research #R1054), following manufacturer’s instructions.
Total RNA samples for each time point and condition were prepared in three biological replicates as described above. A NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to determine RNA concentration and quality; all samples passed quality assessment. PolyA enrichment and library preparation was performed using the KAPA mRNA HyperPrep Kit (Kapa Biosystems #8098115702) according to the manufacturer’s protocols. Briefly, 500 ng of RNA was used as input, and single-index adapters (Kapa Biosystems #08005699001) were added at a final concentration of 10 nM. Purified, adapter-ligated library was amplified for a total of 11 cycles following the manufacturer’s protocol. The final libraries were pooled and sequenced on an Illumina NovaSeq 6000 (University of Colorado Genomics Core) as 150 bp paired-end reads.
Total RNA samples for each time point and condition were prepared in three biological replicates as described above. A NanoDrop One spectrophotometer (Thermo Fisher Scientific) was used to determine RNA concentration and quality; all samples passed quality assessment. PolyA enrichment and library preparation was performed using the KAPA mRNA HyperPrep Kit (Kapa Biosystems #8098115702) according to the manufacturer’s protocols. Briefly, 500 ng of RNA was used as input, and single-index adapters (Kapa Biosystems #08005699001) were added at a final concentration of 10 nM. Purified, adapter-ligated library was amplified for a total of 11 cycles following the manufacturer’s protocol. The final libraries were pooled and sequenced on an Illumina NovaSeq 6000 (University of Colorado Genomics Core) as 150 bp paired-end reads.
7
Comprehensive Cellular RNA Extraction and Analysis
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Cellular total RNA extraction was carried out using RNA Lysis Buffer or DNA/RNA Shield (Zymo Research, Irvine, CA, USA) were subjected to the using Quick‐RNA™ Microprep Kit (Zymo Research, Irvine, CA, USA). The extracted total cellular RNA was applied to cDNA synthesis with qScript cDNA SuperMix (Quantabio, Beverly, MA) for 2‐step RT‐qPCR analysis with Applied Biosystems™ PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Foster city, CA, USA), or subjected to a probe‐based 1‐step RT‐qPCR with TaqMan Fast Virus 1‐Step Master Mix for the detection of HBV genome. The primer and probe sequences are summarized in Table S3 . Poly‐A‐based mRNA sequencing was carried out cDNA library synthesized with NEBNext Ultra™ II RNA Library Prep Kit for Illumina (New England BioLabs, UK) using Illumina NovaSeq 6000 Sequencing System.
8
Studying Necrotic Supernatant Effects
9
Gene Expression Analysis of VICs
10
Temporal Transcriptome of Mosquitoes
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Mosquitoes that emerged over a 24-h period were either directly collected as 0- to 1-day-old adults, or isolated for later collection time points of 1- to 2-day-old and 2- to 3-day-old adults. For each time point, five mosquitoes were homogenized in 300 µl RNA lysis buffer (Zymo Research) and stored at −80 °C until RNA isolation. For RNA isolation, equal volumes of homogenate from each time point were combined to represent 0- to 3-day-old mosquitoes. These steps were performed in biological triplicates for males and virgin nonblood-feed females of both An. stephensi and Ae. aegypti. Illumina paired-end libraries for the resulting samples were prepared using the manufacturer’s specific protocol. The libraries were then sequenced using Illumina HiSeq. The resulting samples have been submitted to the NCBI SRA under the accession SRP047470 and SRP055921.
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