For DcAQP transcript fragment evaluation by semi-quantitative RT-PCR, 2 h after the second dsRNA application, the nymphs were washed with RNase-free water to removed dsRNA-DcAQP residues and were processed. A primer pair (DcAQPSRT F-R) was designed to be inside the dsRNA-DcAQP trigger sequence for semi-quantitative RT-PCR analysis, which produced an amplimer of 234 nucleotides. Then, total RNAs were extracted in TRIzol® Reagent (Invitrogen) from untreated eggs, nymphs in first, second, third, combined fourth-fifth stages, and adults using a Direct-zolTM RNA MiniPrep kit (Zymo Research, Irvine, CA, USA). Total RNA (100 ng) from each sample was used to amplify the transcript fragments of DcAQP, and actin was used as an internal control (GenBank Accession number: DQ675553, Reference Primer ActRT F-R) with the Affinity Script One-Step RT-PCR kit (Agilent). Reactions were subjected to the thermal program: 45 °C for 5 min, 92 °C for 1 min; 30 cycles of 92 °C for 20 s, 57 °C for 20 s and 72 °C for 30 s; followed by maintenance at 72 °C for 3 min. PCR products were analyzed on a 1.5% agarose gel (Thermo Fisher Scientific) and visualized by GelRed staining (Biotium, Fremont, CA, USA).
Affinityscript one step rt pcr kit
Manufactured by Agilent Technologies
Sourced in United States
The AffinityScript One-Step RT-PCR Kit is a laboratory equipment product developed by Agilent Technologies. It is designed for performing reverse transcription and polymerase chain reaction (RT-PCR) in a single step, enabling the conversion of RNA to cDNA and subsequent amplification of the target sequence.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Direct zoltm rna miniprep kit, by Zymo Research (1 mentions)
Gelred staining, by Biotium (1 mentions)
Ribopure kit, by Thermo Fisher Scientific (1 mentions)
Feature extraction, by Agilent Technologies (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Rneasy mini column, by Qiagen (1 mentions)
Gel documentation system, by Bio-Rad (1 mentions)
Hotstartaq master mix kit, by Qiagen (1 mentions)
Sybr safe dna gel stain, by Bio-Rad (1 mentions)
Superscript 3 one step rt pcr kit, by Thermo Fisher Scientific (1 mentions)
Platinum taq, by Thermo Fisher Scientific (1 mentions)
Qiaxcel advanced, by Qiagen (1 mentions)
Hotstartaq dna polymerase kit, by Qiagen (1 mentions)
Qiaxcel screengel software, by Qiagen (1 mentions)
8 protocols using affinityscript one step rt pcr kit
1
Semi-quantitative RT-PCR analysis of DcAQP
2
RNA Extraction and qRT-PCR Analysis
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Total RNA from RAW 264.7 or 3T3L1 cells was extracted by using RNeasy Mini Kit (Qiagen). cDNA was synthesized from RNA using AffinityScript One-Step RT-PCR Kit (Agilent, Santa Clara, USA) as per the manufacturer's protocol. Briefly, 5 µg of total RNA was mixed with the buffer containing Affinity Script reverse transcriptase and polyT primer. The mixture was kept in the thermo cycler at 45°C for 30 min to synthesize cDNA. Then the temperature was raised to 92°C for 1 min to deactivate the enzyme.
qRT-PCR reaction was set in a 96-well PCR plate. The template, required primer, buffer and SYBR green along with DNA polymerase were added in PCR plate as per the manufacturer's protocol. The plate was then kept in the q-RT-PCR machine (Mx3005P, Stratagene, La Jolla, USA) and the machine was programmed as follows: 2 min at 92°C to activate DNA polymerase for 1 cycle, 15 s at 92°C for melting and 1 min at 60°C for primer annealing along with extension of the chain and detection of the florescence for 40 cycles, then a program to find out the melting temperature of each product. Cycle threshold values were noted and fold changes of the desired genes were calculated with respect to the control after normalizing with internal control gene β-ACTIN. The qRT-PCR reactions were set up as three technical replicates along with no template control and no primer control.
qRT-PCR reaction was set in a 96-well PCR plate. The template, required primer, buffer and SYBR green along with DNA polymerase were added in PCR plate as per the manufacturer's protocol. The plate was then kept in the q-RT-PCR machine (Mx3005P, Stratagene, La Jolla, USA) and the machine was programmed as follows: 2 min at 92°C to activate DNA polymerase for 1 cycle, 15 s at 92°C for melting and 1 min at 60°C for primer annealing along with extension of the chain and detection of the florescence for 40 cycles, then a program to find out the melting temperature of each product. Cycle threshold values were noted and fold changes of the desired genes were calculated with respect to the control after normalizing with internal control gene β-ACTIN. The qRT-PCR reactions were set up as three technical replicates along with no template control and no primer control.
3
Reverse Transcription of mRNA to cDNA
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Following the manufacturer’s protocol, we converted the mRNA into cDNA from total RNA using AffinityScript One-Step RT-PCR Kit (Cat# 600559, Agilent, Santa Clara, CA, USA).
4
Gene Expression Profiling of shB7-H3 RKO Cells
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The Agilent human gene expression profiling 4×44K chip was utilized (Agilent, Santa Clara, CA, USA). shB7-H3 RKO and control RKO cells were collected for total RNA extraction with Ribo Pure Kit (AM1924, Thermo Fisher Scientific). Next, total RNA was obtained with RNeasy ® Mini Kit (74106, QIAGEN, Tegelen, Netherlands) to achieve a ratio of A260/A280 close to 2.0. Then, complementary RNA (cRNA) was synthesized with AffinityScript One-Step RT-PCR Kit (600188, Agilent) and labeled with Cy3-CTP (ab97170, Abcam). After another purification step, the concentration, fluorescence, and labeling efficiency of the cRNA were detected with NanoDrop2000C. For the microarray assay, cRNA sample fragmentation and chip hybridization were then conducted; after 3 washes, the microarrays were scanned with an Agilent high resolution scanner, Type C. The data were analyzed by Agilent Feature Extraction. Genes with P<0.05 were considered differentially expressed genes (DEGs).
5
Colon Tissue RNA Extraction and cDNA Synthesis
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RNA was extracted from the colon tissue by using RNeasy mini kit (Cat# 74104, Qiagen, Germany) following the manufacturer's protocol. 20-23 mg of tissue was processed using liquid nitrogen followed by homogenization in 700 μl of RLT buffer. An equal volume of 70% ethanol was added and mixed well. The solution was centrifuged at 8000g for 5 minutes at room temperature. The clear solution containing lysate was passed through RNeasy mini column (Qiagen, Germany), which leads to the binding of RNA to the column. The column was washed using 700 μl RW1 buffer and next with 500 μl of RPE buffer. RNA was eluted using 30 μl of nuclease-free water. RNA was quantified using NanoDrop 2000 (ThermoFisher Scientific, Columbus, OH, USA). cDNA preparation: cDNA was synthesized by using Affinity Script One-Step RT-PCR Kit (Cat# 600559, Agilent, Santa Clara, CA, USA). RNA was mixed with random nonamer primers, certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was not this version posted April 22, 2020. ; https://doi.org/10.1101/516898 doi: bioRxiv preprint 6 Taq polymerase, and NT buffer. The mixture was kept at 45 °C for 30 min for the synthesis of cDNA and temperature increased to 92 °C for deactivating the enzyme.
The copyright holder for this preprint (which was not this version posted April 22, 2020. ; https://doi.org/10.1101/516898 doi: bioRxiv preprint 6 Taq polymerase, and NT buffer. The mixture was kept at 45 °C for 30 min for the synthesis of cDNA and temperature increased to 92 °C for deactivating the enzyme.
6
BVDV Genotyping Protocol Using Nested-Multiplex PCR
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The viral RNA from sera samples was extracted using the Viral Nucleic Acid Kit II (Geneaid®, Geneaid Biotech Ltd., Taiwan) as described by the manufacturer. The extracted RNA was subjected to the nested-multiplex PCR BVD test followed by agarose gel resolution to genotype the BVDV.
The specific NS5B primers used for external reaction and nested-multiplex genotyping are listed asTable-1 [16 (link)]. The first step external reverse transcriptase reactions were performed using the AffinityScript One-Step RT-PCR Kit (Agilent, Santa Clara, CA, USA) as specified by the manufacturer. The thermal conditions for the external reactions were as follows: 45°C for 30 min of reverse transcription, 92°C for 1 min of initial denaturing followed by 40 cycles of 92°C for 20 s, 50°C for 20 s, 68°C for 45 s, and final elongation at 68°C for 5 min. The second stage of the multiplex PCR genotyping was done using HotStar Taq Mastermix Kit (Qiagen®, Hilden, Germany). The reaction was heated to 95°C for 15 min, followed by 35 cycles of 94°C for 40 s, 56°C for 40 s, 72°C for 40 s, with a final elongation step of 72°C for 7 min.
BVDV-1 strain Singer was used as a positive control for genotype-1 in this study. All the PCR products were separated on a 1.5% agarose gel, stained with SYBR® Safe DNA gel stain, and visualized using Gel Documentation Systems from Bio-Rad.
The specific NS5B primers used for external reaction and nested-multiplex genotyping are listed as
BVDV-1 strain Singer was used as a positive control for genotype-1 in this study. All the PCR products were separated on a 1.5% agarose gel, stained with SYBR® Safe DNA gel stain, and visualized using Gel Documentation Systems from Bio-Rad.
7
RNA Extraction and Viral Gene Characterization
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RNA was extracted using TRIzol reagent (invitrogen®) as described in previous studies [20 (link)]. Brain samples were homogenized in 1 mL of TRIzol reagent and 0.2 mL of chloroform. After centrifugation (11,000 rpm during 15 min), the supernatant was mixed with 0.5ml of isopropanol. After a second centrifugation (11,000 rpm during 10 min), the collected supernatant was discarded, and the pellet was washed using 1 mL of ethanol. The tubes were then dried and eluted in 50 µL of RNase free water. In the case of wild canids, we characterized the partial Nucleoprotein gene with a hemi-nested PCR protocol using the SuperScript III One step RT-PCR kit (invitrogen) for the first step and the platinum Taq (Invitrogen) in the second step, following the manufacturer’s instructions. A partial sequence of the phosphoprotein gene was characterized for both wild canids and domestic dogs with in one step using an Affinity Script One-Step RT-PCR kit (Agilent) as recommended by the manufacturer. The primers used for both analyses are presented in Table 2 .
8
Sensitive BCR-ABL1 Detection in Rare Cells
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FACS-purified subfractions containing limited cell numbers (as low as one cell) were analyzed for BCR-ABL1 positivity by direct nested reverse transcription PCR. Primers were used according to a previously published method [21 (link)]. Initially, cells were lysed using guanidine thiocyanate, followed by three quick freeze-thaw cycles at −80°C. The first-step of the nested RT-PCR was performed using the AffinityScript One-Step RT-PCR Kit (Agilent Technologies, USA) and included a single-tube reverse transcription and the first PCR round [22 (link)]. The resulting PCR product was purified with a mixture of Exo I and FastAP Thermosensitive Alkaline Phosphatase at a ratio of 1:2 (Fermentas – Thermo Fisher Scientific, USA). The purified PCR product was diluted 100× in RNase-free water. For a second amplification, the HotStar Taq DNA Polymerase Kit (Qiagen, Germany) was used. Amplification products from both PCR rounds were visualized using a QX DNA Screening Kit on a QIAxcel Advanced instrument and analyzed using QIAxcel ScreenGel software (all Qiagen, Germany).
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