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High pure rna isolation kit

Manufactured by Roche
Sourced in Germany, Switzerland, United States, Japan, United Kingdom, France, Italy, Australia, Canada, Belgium, Hong Kong, Spain

The High Pure RNA Isolation Kit is a laboratory product designed for the rapid and efficient isolation of high-quality total RNA from a variety of sample types, including cells, tissues, and body fluids. The kit utilizes a specially-developed silica-based membrane technology to capture and purify RNA, while removing contaminants and inhibitors. The isolated RNA can be used in a wide range of downstream applications, such as qRT-PCR, Northern blotting, and microarray analysis.

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1 302 protocols using high pure rna isolation kit

1

Influenza A H5N1 Genome Amplification

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For the human H5N1 samples 200 µl of clinical sample was added to 400 µl lysis-binding buffer (High pure RNA isolation kit, Roche) and RNA extraction was subsequently performed using the High pure RNA isolation kit (Roche) with an on-column DNase treatment according to the manufacturer's protocol. Total RNA was eluted in a volume of 50 µl elution buffer (Roche) and directly used for reverse transcription and amplification. cDNA was synthesized using the Uni12M primer (AGCRAAAGCAGG) [31 (link)] and the Superscript III First-Strand Synthesis System according to the manufacturer's protocol (Invitrogen), followed by amplification of the whole genome in an overlapping amplicon approach using degenerative primer sets (primer sequences available upon request) [18 (link),32 (link)]. PCR reactions were performed using Platinum Taq DNA Polymerase High Fidelity (Invitrogen). Thermal cycling conditions were: denaturation at 95°C for 5 min; 40 cycles of 95°C for 30 s, 50°C for 30 s, 68°C for 1 min; and final extension at 68°C for 5 min.
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2

RNA Isolation from Cytotoxic Compounds

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The High Pure RNA
Isolation Kit (Roche Life Sciences, Germany) was used for the purification
of total RNA from cultured cells. The IC50 doses of the
two most cytotoxic derivatives-compounds 23 (4.7 μM)
and 27 (10.9 μM) were administered to MCF-7 cells
and incubated for 72 h. Vincristine (1 μM) and untreated MCF-7
cells were used as the positive and negative controls, respectively.
RNA was isolated from cells using a High Pure RNA Isolation Kit (Roche
Life Sciences, Germany) according to the manufacturer’s instructions.
The concentration and purity of RNA samples were determined using
an AlphaSpec (α Innotech) spectrophotometer. RNA samples with
an optical density (OD260/OD280) ratio of 1.8–2.0,
which denotes good purity, were selected based on their absorbance
readings.
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3

Isolation and Characterization of RNA

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Cultured cells were rinsed twice with phosphate buffered saline (PBS, Sigma-Aldrich Co. St. Louis, MO, USA) and collected using Lysis/Binding Buffer of High Pure RNA isolation kits (Roche Diagnostics, Basel, Switzerland). RNA was extracted from cells using High Pure RNA isolation kits (Roche Diagnostics). Total RNA from human adult skeletal and heart muscles was obtained from a human total RNA Master Panel II (Clontech Laboratories, Inc., Mountain View, CA, USA). cDNA was synthesized from 0.5 µg of each total RNA using random primers as described [25 (link)].
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4

RNA Extraction and qRT-PCR Analysis

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A High Pure RNA Isolation Kit (Roche Ltd., Mannheim, Germany) was used to extract total RNAs from the cells according to the manufacturer’s protocol [16,17] . One microgram of total RNAs was reverse-transcripted to produce first strand cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan) according to the manufacturer’s protocol [16,17] . The Thermal Cycler Dice Real Time System (TaKaRa, Shiga, Japan) was used for a 2-step reverse transcription polymerase chain reaction (PCR). The mRNA transcripts were quantified by SYBR Premix ExTaq (TaKaRa, Shiga, Japan). HO-1 specific primers 5′-GCTCAAAAAGATTGCCCAGAA-3′ and 5′-TCACATGGCATAAAGCCCTACA-3′ were used. The other primers were purchased from TaKaRa. The amplification conditions included 30 s at 95 °C, a run of 45 cycles at 95 °C for 5 s, and 60 °C for 60 s followed by dissociation for 15 s at 95 °C and 30 s at 60 °C and then 15 s at 95 °C on the Thermal Cycler Dice Real Time System. The Thermal Cycler Dice Real Time System analysis software (TaKaRa, Shiga, Japan) was used to analyze the data. The Ct values (cycle threshold) were calculated by the crossing-point method, and the relative quantities of target mRNA expression levels were measured by the comparison of standard curve. The results for each sample were normalized to ACTB, the housekeeping gene.
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5

Quantifying Gene Expression in Differentiation

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RNA from each condition and the controls was extracted using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland) following the manufacturer’s instructions. RNA was quantified using a nanodrop and 1 µg of RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Relative gene expression was evaluated using 10 ng of cDNA, 250 nM of each primer (Table S1), and by using the Fast SYBR Green Master Mix (Thermo Fisher Scientific) with an annealing temperature set to 60 °C. Melting curves were performed at the end to assess if the primers were amplifying only the correct amplicon. The values were treated following the 2−ΔΔCT method. GAPDH gene expression was used as an endogenous control and the relative expression was calibrated using day 0 of differentiation.
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6

RNA Isolation and Sequencing Protocol

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For RNA sequencing purposes (RNA-seq), three aliquots of the cell line and specimens (tumour core and liver) from seven animals were collected. The RNA purification system PeqGold TriFast (Peqlab, Erlangen, Germany) was used to isolate RNA from metastatic liver tissue. Briefly, specimens were defrosted in PeqGold TriFast (1 ml/100 mg tissue) and then homogenized using TissueLyser LT (Qiagen, Hilden, Germany) at 50 Hz. Total RNA was isolated according to the manufacturer’s instructions and stored at −80 °C. In addition, the High Pure RNA Isolation Kit (Roche, Grenzach-Wyhlen, Germany) was used to isolate RNA from CMT-93 cells according to the manufacturer’s recommendations. The quantity and integrity of the isolated RNA was assessed in a NanoDrop ND − 1000 spectrophotometer, version 3.5.2 (Peqlab, Erlangen, Germany), using the 260 nm/280 nm absorbance ratio and was further analysed with an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, California, USA) as a quality check. RNA-seq was performed at the Transcriptome and Genome Analysis Laboratory in Goettingen, Germany, using an Illumina HiSeq2000 sequencer (Illumina, Inc., San Diego, California, USA).
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7

RNA Extraction and Microarray Analysis

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Total RNAs were extracted using the High Pure RNA Isolation Kit (Roche Diagnostics) hybridized to Agilent SurePrint G3 Mouse GE 8x60K microarrays according to the manufacturers’ protocol.
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8

Mouse Transcriptome Profiling via Microarray

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Total RNAs were extracted using the High Pure RNA Isolation Kit (Roche Diagnostics) hybridized to Agilent SurePrint G3 Mouse GE 8×60K microarrays according to the manufacturers’ protocol.
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9

Purification of Undifferentiated Spermatogonia

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Undifferentiated spermatogonia from 9-dpp males were purified using the method described previously (Garcia and Hofmann, 2012 ). Note this method was also used to prepare testicular germ cells for FACS analysis. For 1-dpp testes, male animals were sacrificed and pairs of testes removed. Tubules were dissociated by collagenase. Single-cell suspensions were prepared by incubation with Accutase (Sigma) and passing through a cell strainer. Germ cells were purified on either a FACSAria III or Mo-Flo cell sorter, with purities for both wild-type and mutant germ cells at >98%. DNA was prepared using the QIAamp Micro Kit (QIAGEN), and RNA was prepared using the High Pure RNA Isolation Kit (Roche). All animals used for this study were covered by a Home Office Project License under The Animals (Scientific Procedures) Act 1986 to S.K.T.O. This research study was also approved by the UCL Research Ethics Committee. Animals were humanely sacrificed at a designated establishment by cervical dislocation.
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10

Purification of Undifferentiated Spermatogonia

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Undifferentiated spermatogonia from 9-dpp males were purified using the method described previously (Garcia and Hofmann, 2012 ). Note this method was also used to prepare testicular germ cells for FACS analysis. For 1-dpp testes, male animals were sacrificed and pairs of testes removed. Tubules were dissociated by collagenase. Single-cell suspensions were prepared by incubation with Accutase (Sigma) and passing through a cell strainer. Germ cells were purified on either a FACSAria III or Mo-Flo cell sorter, with purities for both wild-type and mutant germ cells at >98%. DNA was prepared using the QIAamp Micro Kit (QIAGEN), and RNA was prepared using the High Pure RNA Isolation Kit (Roche). All animals used for this study were covered by a Home Office Project License under The Animals (Scientific Procedures) Act 1986 to S.K.T.O. This research study was also approved by the UCL Research Ethics Committee. Animals were humanely sacrificed at a designated establishment by cervical dislocation.
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