B. malayi FR3 RNA was isolated using TRIzol (Zymo Research, Irvine, CA, USA) with tissue homogenization followed by purification with a PureLink RNA Mini column (Ambion, Austin, TX, USA) (21 (link)). Publicly available (SRR15923920) D. ananassae ONT direct RNA sequencing data was used (Supplementary Table 1 ) that was sequenced from RNA isolated from whole flies homogenized in liquid nitrogen using QIAzol and chloroform (22 (link)). C. albicans RNA was isolated using TRIzol (Zymo Research, Irvine, CA, USA) with bead beating and a PureLink RNA Mini column (Ambion, Austin, TX, USA). E. coli RNA was isolated using an RNEasy column (Qiagen, Germantown, MD, USA) followed by polyadenylation with an E. coli poly(A) polymerase (NEB, Ipswich, MA, USA). Total RNA was extracted from SINV-infected JW18 cells using TRIzol reagent (Invitrogen, Waltham, MA, USA) followed by RQ1 RNase-free DNase (NEB, Ipswich, MA, USA) treatment using manufacturer’s protocol. SINV IVT RNA was generated by SP6-driven transcription with MEGAscript (Thermo Fisher Scientific Inc., Waltham, MA, USA) and the TE12 BC 4.10 plasmid, followed by lithium chloride precipitation.
Purelink rna mini column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Purelink RNA Mini columns are designed for the rapid purification of high-quality RNA from a variety of sample types. The columns utilize a silica-based membrane to efficiently capture and purify RNA, which can then be eluted for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Trizol, by Zymo Research (3 mentions)
Mastercycler realplex2 system, by Eppendorf (2 mentions)
Turbo dnase, by Thermo Fisher Scientific (2 mentions)
Sybr green fluorescent dye, by Thermo Fisher Scientific (2 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Quantstudio 5 qpcr machine, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Qiagen (2 mentions)
Tissuelyser, by Qiagen (2 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Oligo dt 20 primer, by Thermo Fisher Scientific (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 real time pcr system, by Roche (1 mentions)
Lightcycler 480 sybr green 1 master mix, by Roche (1 mentions)
Prism 7300 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
Rna bee, by AMS Biotechnology (1 mentions)
Taqman preamp master mix, by Thermo Fisher Scientific (1 mentions)
14 protocols using purelink rna mini column
1
RNA Isolation Protocols Across Species
2
Quantitative RNA Analysis of Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested in mid-log phase, resuspended in 1 ml of TRIzol (Invitrogen), and incubated for 5 min at room temperature before the addition of 200 μl of chloroform. Samples were centrifuged at 13,000 relative centrifugal force (rcf) for 10 min. The aqueous phase was mixed with 200 μl of 100% ethanol, and the mixture was transferred to Purelink RNA Mini columns (Invitrogen) for purification following the manufacturer’s instructions. RNA was treated with Turbo DNase (Invitrogen) according to the manufacturer’s instructions. The KAPA SYBER FAST One-Step quantitative reverse transcription-PCR (qRT-PCR) kit (Kapa Biosystems) was used to measure mRNA abundance on the Eppendorf Mastercycler RealPlex2 system. Relative gene expression changes were calculated using the Livak method (55 (link)). The primers used for the detection of hcp-1 and ctxA are listed in Table S5 . The reference gene used was gyrA.
3
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and resuspended in 1 mL of TRIzol (Invitrogen) and incubated for 5 min at room temperature before adding 200 µL of chloroform. Samples were centrifuged at 13,000 relative centrifugal field (rcf) for 10 min. The aqueous phase was mixed with 200 µL of 100% ethanol and transferred to Purelink RNA Mini columns (Invitrogen) for purification following the manufacturer’s instructions. RNA was treated with TURBO DNase (Invitrogen) according to the manufacturer’s instructions. The KAPA SYBER FAST One-Step qRT-PCR Kit (Kapa Biosystems) was used to measure mRNA abundance on the Eppendorf Mastercycler RealPlex2 system. Relative gene expression changes were calculated using the Livak method (37 (link)).
4
Isolating High-Quality Total RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from 0.5 to 1.0 g of tissue was isolated with TRIzol reagent (Ambion) and puri ed with simultaneous treatment with DNAase I on PureLink RNA Mini columns (Invitrogen). RNA quality was determined on a BA2100 Bioanalyzer using an RNA Nano Kit.
5
Robust RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol (Invitrogen) combined with PureLink RNA Mini columns (Thermo Fisher, 12183025) for tissue and SVC cells or by PicoPure RNA Isolation Kit (Applied Biosystems, KIT0204) for 384-well plate populations and adipocytes. Prior to the addition of chloroform, all tissue and primary adipocytes in TRIzol included an extra spin at max speed for 10 minutes at RT, then TRIzol was removed from below the lipid layer to avoid lipid contamination disrupting the subsequent phase separation with chloroform. Chloroform was added to the lipid-free TRIzol, spun for 15 mins at 12,000 × g and the aqueous layer was removed and added to columns. mRNA was quantified using a Nanodrop and reverse transcribed to cDNA using the ABI High-Capacity cDNA Synthesis kit (ABI, 4368813). Real-time PCR was performed on a QuantStudio5 qPCR machine using SYBR green fluorescent dye (Applied Biosystems). Fold changes were calculated using the ddCT method, with TATA binding Protein (Tbp) mRNA serving as a normalization control.
6
Constructing 5'UTR Reporter Constructs
7
RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol (Invitrogen) combined with PureLink RNA Mini columns (Thermo Fisher, 12183025) for tissue and SVC cells or by PicoPure RNA Isolation Kit (Applied Biosystems, KIT0204) for 384-well plate populations and adipocytes. Prior to the addition of chloroform, all tissue and primary adipocytes in TRIzol included an extra spin at max speed for 10 min at RT, then TRIzol was removed from below the lipid layer to avoid lipid contamination disrupting the subsequent phase separation with chloroform. Chloroform was added to the lipid-free TRIzol, spun for 15 min at 12,000 x g and the aqueous layer was removed and added to columns. mRNA was quantified using a Nanodrop and reverse transcribed to cDNA using the ABI High-Capacity cDNA Synthesis kit (ABI, 4368813). Real-time PCR was performed on a QuantStudio5 qPCR machine using SYBR green fluorescent dye (Applied Biosystems). Fold changes were calculated using the ddCT method, with TATA binding Protein (Tbp) mRNA serving as a normalization control.
8
Quantitative Analysis of Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted using TRIzol reagent (Life Technologies) and on column DNase digestion was performed during purification of total RNA using PureLink RNA Mini columns (Life Technologies) according to manufacturer’s instructions. Subsequently, cDNA was prepared by reverse transcription using oligo-(dT)20 primer (Life Technologies) and SuperScript II Reverse Transcriptase (Life Technologies) for further PCR analysis.
qPCR for SOX5, BLIMP1 and ribosomal protein, large, P0 (RPLP0) genes was performed by a LightCycler 480 Real-Time PCR System (Roche Diagnostics GmbH) and based on SYBR Green I detection (LightCycler 480 SYBR Green I Master mix, Roche Diagnostics GmbH) using the following conditions: activation at 95°C for 5 min and 50 cycles of amplification at 95°C for 10 s, 63°C for 20 s and 72°C for 30 s., followed by subsequent melting curve analysis. RPLP0 gene was used as an endogenous internal control in samples. PCR primer sequences are provided in thetable S1 .
qPCR for SOX5, BLIMP1 and ribosomal protein, large, P0 (RPLP0) genes was performed by a LightCycler 480 Real-Time PCR System (Roche Diagnostics GmbH) and based on SYBR Green I detection (LightCycler 480 SYBR Green I Master mix, Roche Diagnostics GmbH) using the following conditions: activation at 95°C for 5 min and 50 cycles of amplification at 95°C for 10 s, 63°C for 20 s and 72°C for 30 s., followed by subsequent melting curve analysis. RPLP0 gene was used as an endogenous internal control in samples. PCR primer sequences are provided in the
9
Gene Expression Analysis in Skeletal Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cellular RNA was isolated from ~30 mg of pulverized gastrocnemius muscle using a FastRNA Green Kit (MP Bio, Santa Ana, CA, USA) and RNA-containing homogenates were further purified using PureLink RNA mini columns (Life Technologies, Carlsbad, CA, USA). Quantification of RNA was performed on each sample using spectrophotometry by measuring the absorbance at 260 nm using a Nanodrop 1000 (Thermo Scientific, Rockford, IL, USA). RNA purity was determined by a 260:280 nm ratio of ≥2.0 and the absence of organic contamination such as phenol and carbohydrates was verified by a 230:260 nm ratio of ≥1.8. RNA (1 μg), which was reverse transcribed (#4368814; Life Technologies, Carlsbad, CA, USA) and 25 ng of cDNA per reaction was used for real-time PCR with TaqMan gene expression assays (#4331182; Life Technologies, Carlsbad, CA, USA). Levels of MAFbx (Mm00499523_m1), MuRF-1 (Mm01185221_m1), and myostatin (Mm01254559_m1) mRNA were analyzed and normalized to TATA box binding protein endogenous control reference mRNA (Tbp; Mm01277042_m1) using a Prism 7300 real-time PCR system (Life Technologies, Carlsbad, CA, USA). Target mRNA levels were calculated using the ∆∆Ct method and expressed relative to control samples.
10
Brain RNA Isolation and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To extract total RNA, an aliquot of brain homogenate was mixed with RNABee (Amsbio) reagent, and the solution was incubated on ice with chloroform. The resultant aqueous layer was removed, and RNA was isolated using PureLink RNA Mini Columns (Life Technologies) according to manufacturer’s protocol. Contaminating genomic DNA was removed, and complementary DNA (cDNA) was reverse-transcribed with High Capacity RNA to cDNA conversion kit (Life Technologies) from equal concentrations of RNA across samples. Pre-amplification (14 cycles) was subsequently performed using TaqMan PreAmp Master Mix (Applied Biosystems) with desired targets. Pre-amplified, diluted cDNA was then used to run quantitative, real-time RT-PCR on a StepOne Plus Real-Time PCR System with desired TaqMan probes (all from Applied Biosystems; probe numbers are available upon request). Expression levels of mRNA were determined relative to housekeeping genes GAPDH and 18S RNA, and data were normalized to a wildtype calibrator sample. Statistics were performed at the relative (delta Ct) level [19 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!