Total RNA was isolated using NucleoSpin® RNA XS (Macherey-Nagel) from in vitro grown oocytes after 10 days of follicle culture or from oocytes obtained directly from adult (C57BL × CBA) F1 females (7-11 weeks old). A total of 50 oocytes with an intact nucleus per sample were used and 3 samples per group were collected. RNA was extracted using NucleoSpin RNA XS (Macherey-Nagel). A cDNA library was prepared using SMARTer® Ultra™Low Input RNA for Sequencing (Clonetech Laboratories) and the samples were processed by BGI Tech Solutions. The cDNA product was synthesized and amplified using SMARTer PCR cDNA Synthesis Kit (Clontech) from the total RNA (10 ng) of sample. The cDNA was fragment by Covaris E210 (Covaris) and the median insert length was about 200bp. The paired-end cDNA library was prepared in accordance with Illumina’s protocols with an insert size of 200 bp and sequenced for 100 bp by HiSeq2000 (Illumina).
Nucleospin rna xs
Manufactured by Macherey-Nagel
Sourced in Germany, France, United States
The NucleoSpin RNA XS is a compact RNA isolation kit designed for the purification of total RNA from small samples. It utilizes silica-membrane technology to efficiently capture and purify RNA from a variety of sample types, including cultured cells, tissues, and bacteria.
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Lab products found in correlation
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (9 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (7 mentions)
Cfx96 real time pcr system, by Bio-Rad (5 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Verso cdna synthesis kit, by Thermo Fisher Scientific (4 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (4 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (4 mentions)
Lightcycler 480, by Roche (3 mentions)
Iscript cdna synthesis kit, by Bio-Rad (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Sybr green system, by Bio-Rad (3 mentions)
Iscript reverse transcription supermix, by Bio-Rad (3 mentions)
Xs plus kit, by Macherey-Nagel (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (3 mentions)
Maxima h minus first strand cdna synthesis kit, by Thermo Fisher Scientific (3 mentions)
Taqman pcr master mix amplitaq gold with geneamp kit, by Thermo Fisher Scientific (2 mentions)
Rotor gene rg 3000 machine, by Qiagen (2 mentions)
98 protocols using nucleospin rna xs
1
RNA-seq of Mouse Oocyte Transcriptome
2
RNA-seq of Mouse Oocyte Transcriptome
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Total RNA was isolated using NucleoSpin® RNA XS (Macherey-Nagel) from in vitro grown oocytes after 10 days of follicle culture or from oocytes obtained directly from adult (C57BL × CBA) F1 females (7-11 weeks old). A total of 50 oocytes with an intact nucleus per sample were used and 3 samples per group were collected. RNA was extracted using NucleoSpin RNA XS (Macherey-Nagel). A cDNA library was prepared using SMARTer® Ultra™Low Input RNA for Sequencing (Clonetech Laboratories) and the samples were processed by BGI Tech Solutions. The cDNA product was synthesized and amplified using SMARTer PCR cDNA Synthesis Kit (Clontech) from the total RNA (10 ng) of sample. The cDNA was fragment by Covaris E210 (Covaris) and the median insert length was about 200bp. The paired-end cDNA library was prepared in accordance with Illumina’s protocols with an insert size of 200 bp and sequenced for 100 bp by HiSeq2000 (Illumina).
3
Molecular Characterization of Anopheles aquasalis SG7
4
Transcriptomic Analysis of Abiotic Stress in MEAM1
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As we considered the 24°C temperature regime a more abiotic stressful environment for MEAM1 (Butler et al. 1983 ; Nava-Camberos et al. 2001 ; Tsueda & Tsuchida 2011 ; Zidon et al. 2016 (link)), newly emerged adults (1–7 days old) for transcriptomic analysis were collected only from the 24°C-treated cages, separated by sex (males and females) and pooled by treatment (50 individuals in each biological replicate). The C→C and hP→C treatments had five female replicas, the hP→hP treatment had four female replicas, and the C→hP treatment had three female replicas. The female samples were homogenized immediately upon collection with the first buffer (NucleoSpin RNA XS, Macherey-Nagel) using 1-mm sterilized zirconia beads and two pulses of 60 seconds at 5,000 rpm of a bead-beater (Minilys, Bertin Technologies). The male samples were saved for analyzing the epigenetic effects (DNA methylation) on gene expression (currently conducted). Total RNA was extracted from the homogenates according to the manufacturer’s instructions (NucleoSpin RNA XS, Macherey-Nagel). Library construction and sequencing were performed by Macrogen Inc. TruSeq RNA Library v2 libraries were constructed and sequenced on a NovaSeq 6,000 platform. On average, it produced approximately 30 million 100-bp pair-ended reads per sample (biological replicate) (supplementary tables S4 and S5 , Supplementary material online).
5
Sorting and Analyzing Scrib Mutant Cells
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Eye imaginal discs containing scrib and wild-type cells were dissected in PBS and the discs were collected in Schneider's medium (Thermo Fisher Scientific). The scrib and wild-type cells from 40 eye imaginal discs were dissociated with TrypLEÔ Select Enzyme (Thermo Fisher Scientific). The dissociated cells were directly collected into lysis buffer (NucleoSpin RNA XS, MACHEREY-NAGEL) using FACS Aria II cell sorter (BD Biosciences) according to GFP or RFP fluorescent.
The detailed protocol is described in Khan et al. (2016) .
qRT-PCR Total RNA was extracted from homogenized wandering L3 larval brains using NucleoSpin RNA XS (MACHEREY-NAGEL) and prepared for reverse transcription using SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Quantitative real-time PCR was performed using THUNDERBIRD SYBR qPCR mix (TOYOBO) on a StepOnePlus system (Thermo Fisher Scientific). Analysis was performed with StepOne software (Thermo Fisher Scientific). Reactions were carried out in multiple replicate indicated in the figure legends. All expression levels were normalized to those of alpha-tubulin (Ponton et al., 2011) . The fold changes in mRNA expression were calculated using the relative quantification standard curve method, and expression values were expressed as fold increase in the average expression compared with control.
The detailed protocol is described in Khan et al. (2016) .
qRT-PCR Total RNA was extracted from homogenized wandering L3 larval brains using NucleoSpin RNA XS (MACHEREY-NAGEL) and prepared for reverse transcription using SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Quantitative real-time PCR was performed using THUNDERBIRD SYBR qPCR mix (TOYOBO) on a StepOnePlus system (Thermo Fisher Scientific). Analysis was performed with StepOne software (Thermo Fisher Scientific). Reactions were carried out in multiple replicate indicated in the figure legends. All expression levels were normalized to those of alpha-tubulin (Ponton et al., 2011) . The fold changes in mRNA expression were calculated using the relative quantification standard curve method, and expression values were expressed as fold increase in the average expression compared with control.
6
Transcriptomic Analysis of Cumulus Cells
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Three independent experiments with 3–4 independent samples containing 30 CC collected after 6 h IVM (therefore, non-expanded CC) in presence or absence of BPS (10 nM or 1 µM) were used for transcriptomic analysis by real-time polymerase chain reaction (qPCR). Briefly, total RNA was extracted from CCs using Tri reagent (T9424, Cergy Pontoise, France) and treated using XS RNA Nucleospin (Macherey Nagel, France), following the manufacturer’s instructions. Subsequently, the RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France), and RNA integrity was checked by electrophoresis. DNase treatment and reverse transcription were performed on 150 ng total extracted CC RNA using the Maxima First Strand cDNA Synthesis kit (Thermo-Fisher Scientific), according to the manufacturer’s recommendations.
7
Quantitative Analysis of Gene Expression
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Six independent experiments with 3–4 HGC samples collected after 48-h treatment in the presence or absence of BPS (10 nM or 1 µM) were used for transcriptomic analysis using real-time quantitative polymerase chain reaction (qPCR). Briefly, total RNA was extracted from HCG using the TRI reagent and treated using XS RNA Nucleospin (Macherey Nagel, France), following the manufacturer’s instructions. Subsequently, the RNA concentration was determined using a NanoDrop ND-1000 spectrophotometer (Nyxor Biotech, Paris, France), and RNA integrity was checked using electrophoresis. DNAse treatment and reverse transcription (RT) was performed on 150 ng total RNA extracted from CC using the Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific), according to the manufacturer’s recommendations.
qPCR reactions were performed as previously described [59 (link)]. The geometric mean of two housekeeping genes (ribosomal protein L19 [RPL19] and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) was used to normalise gene expression. The relative amounts of gene transcripts (R) were calculated according to the equation:
where E is the primer efficiency (Table 1 ) and Ct the cycle threshold.
qPCR reactions were performed as previously described [59 (link)]. The geometric mean of two housekeeping genes (ribosomal protein L19 [RPL19] and glyceraldehyde 3-phosphate dehydrogenase [GAPDH]) was used to normalise gene expression. The relative amounts of gene transcripts (R) were calculated according to the equation:
where E is the primer efficiency (
8
RNA Extraction and qRT-PCR Analysis
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RNA was prepared using the NucleoSpin RNA XS (Macherey-Nagel, Düren, Germany). First-strand cDNA was prepared from 600 ng RNA using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). cDNA was diluted (1 : 20) and real-time PCR was performed using the Rotor-Gene RG-3000 machine (Corbett Research; Qiagen, Hilden, Germany) and the TaqMan PCR Master Mix (AmpliTaq Gold with GeneAmp kit; Applied Biosystems) in 20 μL reaction volumes. Data analyses were performed with the ddCT method using β-actin as an internal control. Results are represented as fold induction compared to control. TaqMan gene expression probes for mouse genes (Applied Biosystems) are provided in Supplementary Table 2.
9
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from mouse brains, using TRIzol (GRISP, Ref. No GB23.0100; Porto, Portugal) and the RNeasy Mini Kit (Machery-Nagel; Dueren, Germany) from blood using the NucleoSpin® RNA Blood (Machery-Nagel; Dueren, Germany) and from cell suspension using the NucleoSpin® RNA XS (Machery-Nagel, Ref. No 12733391; Dueren, Germany). Total RNA was retrotranscribed to cDNA (Transcriptor First Strand cDNA Synthesis Kit, ThermoFisher LTI, Ref. No 18080-051; Bleiswijk, The Netherlands) for PCR with Power SYBR Green PCR master mix (BioRad, Ref. No 1725124; Madrid, Spain). Transcript number was calculated from the threshold cycle (Ct) of each gene with a 2−ΔΔCT method (relative number), normalized to ArbP0 or GADPH, and expressed as fold induction of animals used as controls. The primers used in this study are listed below in Table 1 .
10
Quantitative Real-Time PCR Analysis
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Total RNA was collected using the NucleoSpin® RNA XS (Macherey–Nagel, Duren, Germany), and cDNA was synthesized using Oligo(dT)20 primers (Invitrogen) and SuperScript® III reverse transcriptase (Invitrogen). DNA was amplified with the Applied Biosystems 7500 Fast real-time PCR system according to the manufacturer’s instructions. Data were processed by using the ΔΔCT method72 (link). Details regarding the use of the primers are described in the Supplementary Methods .
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