Total RNA was extracted from the snap-frozen tumor tissues stored at–80 °C after excision, using the acid phenol-guanidinum extraction method, purified using commercially available sets (RNeasy Mini Kit, Qiagen) and treated with DNAase (Qiagen) [17 (link)]. In order to obtain a high amount of RNA, macrodissection was used in all cases. Specimens were visually assessed by the pathologist to confirm that at least 50 % of tumor cells within the sample and areas with highest content of neoplastic tissue were used for direct RNA extraction. The quantity of RNA was measured using the NanoDrop 1000 (Thermo Scientific). RNA samples’ quality was analysed using 2000 Bioanalyzer (Agilent Technologies), and after capillary electrophoresis the RNA integrity number (RIN) was generated by the software for each specimen.
Dnaase
Manufactured by Qiagen
Sourced in United Kingdom
DNAase is a laboratory product designed for the degradation of DNA. It functions to break down DNA molecules through enzymatic activity, facilitating the removal of DNA from samples or experimental setups.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Oligodtt, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Bioanalyzer 2000, by Agilent Technologies (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Cfx connect light cycler, by Bio-Rad (1 mentions)
Iscript, by Bio-Rad (1 mentions)
Kapa sybr fast, by Roche (1 mentions)
Cfx manager software v3, by Thermo Fisher Scientific (1 mentions)
6 protocols using dnaase
1
RNA Extraction from Frozen Tumor Tissues
2
Quantitative RT-PCR Gene Expression Analysis
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Total RNA was isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol. DNAase (Qiagen) treatment was performed to remove genomic DNA. cDNA was synthesized using iScript (Biorad) by using 1 µg of RNA. For qRT-PCR, KAPA SYBR FAST (Kapa Biosystems) was used and the reaction was run in Connect CFX light cycler (Biorad). Primers were designed using qPrimerDepot in such a way that the PCR product spans across exons junctions. Primer sequences are listed in Supplementary Table 1 . Primer specificity was checked using melting curve analysis in CFX manager software v3.1 (Applied Biosystems) and PCR product electrophoresis. Threshold data were analyzed by CFX manager software v3.1 using Comparative Ct relative quantitation method with TBP as an internal control.
3
Mosquito RNA Extraction and Reverse Transcription
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RNA was extracted from 7-10 female mosquitoes in biological triplicate for each experimental group. RNA was extracted from homogenised mosquitoes using a PicoPure RNA isolation kit (Thermo Fisher, UK) following manufacturers’ instructions and treated with DNAase (Qiagen) to remove any DNA contamination. Quality of RNA was checked using a nanodrop spectrophotometer (Nanodrop Technologies UK). 1-4µg of RNA from each experimental set was reversed transcribed using OligoDTT (Invitrogen) and Superscript III (Invitrogen) according to manufacturers’ instructions. The following experimental groups were used: (i) knockdown efficacy for each transcription factor and the GFP control using females 48-hours post RNAi injection and (ii) pathway validation using females 48-hours after they were exposed to 0.05% deltamethrin for 48-hours post-injection for transcription factors and GFP controls.
4
Simultaneous RNA and DNA Extraction from Tumor Tissue
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50–100 mg of tumor tissue was homogenized using a Dounce
homogenizer with 1 ml TRIzol (Ambion); after phase separation by extraction with
0.2 ml chloroform, the aqueous phase was recovered for RNA extraction and the
interphase and phenol-chloroform phase were used for isolation of genomic DNA.
Total RNA and genomic DNA were extracted according to the manufacturer’s
instruction. Briefly, RNA was precipitated by adding an equal volume of
100% Isopropanol with 5 μg/ml linear acrylamide to the aqueous
phase of TRIzol mixture. The RNA pellet was then washed with 75% ethanol
and treated with DNAase (QIAGEN) to remove contaminating DNA. Genomic DNA was
precipitated by adding 0.3 ml 100% ethanol to phenol-chloroform phase of
TRIzol mixture. Then the DNA pellet was washed by citrate/ethanol solution (0.1
M sodium citrate in 10% ethanol, pH8.5). After washing, the DNA pellet
was dissolved in 8 mM NaOH and then adjusted to pH7-8 with HEPES.
homogenizer with 1 ml TRIzol (Ambion); after phase separation by extraction with
0.2 ml chloroform, the aqueous phase was recovered for RNA extraction and the
interphase and phenol-chloroform phase were used for isolation of genomic DNA.
Total RNA and genomic DNA were extracted according to the manufacturer’s
instruction. Briefly, RNA was precipitated by adding an equal volume of
100% Isopropanol with 5 μg/ml linear acrylamide to the aqueous
phase of TRIzol mixture. The RNA pellet was then washed with 75% ethanol
and treated with DNAase (QIAGEN) to remove contaminating DNA. Genomic DNA was
precipitated by adding 0.3 ml 100% ethanol to phenol-chloroform phase of
TRIzol mixture. Then the DNA pellet was washed by citrate/ethanol solution (0.1
M sodium citrate in 10% ethanol, pH8.5). After washing, the DNA pellet
was dissolved in 8 mM NaOH and then adjusted to pH7-8 with HEPES.
5
RNA Extraction and RT-qPCR Analysis of Mosquito Transcription Factors
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RNA was extracted from 7-10 female mosquitoes in biological triplicate for each experimental group. RNA was extracted from homogenised mosquitoes using a PicoPure RNA isolation kit (Thermo Fisher, UK) following manufacturers' instructions and treated with DNAase (Qiagen) to remove any DNA contamination. Quality of RNA was checked using a nanodrop spectrophotometer (Nanodrop Technologies UK). 1-4µg of RNA from each experimental set was reversed transcribed using OligoDTT (Invitrogen) and Superscript III (Invitrogen) according to manufacturers' instructions. The following experimental groups were used: (i) knockdown efficacy for each transcription factor and the GFP control using females 48-hours post RNAi injection and (ii) pathway validation using females 48-hours after they were exposed to 0.05% deltamethrin for 48-hours post-injection for transcription factors and GFP controls.
6
Simultaneous RNA and DNA Extraction from Tumor Tissue
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