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5X All-In-One RT MasterMix

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The 5X All-In-One RT MasterMix is a ready-to-use solution for reverse transcription and subsequent real-time PCR amplification. It contains all the necessary components for both reactions in a single tube.

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285 protocols using 5X All-In-One RT MasterMix

Total RNA was isolated from the root, seedings, leaves, inflorescence, and siliques using a HiPure Plant RNA Mini Kit (Magen, R4151-02) according to the protocol. First-strand cDNA was synthesized from 2 μg of total RNA using 5× All-In-One RT MasterMix (abm, G490). The qRT-PCR was performed using ChamQTM SYBR qPCR Master Mix (Vazyme, Q311-00) to detect the transcript levels of genes. For RT-PCR, the cDNA was amplified with specific primers for CPD, DWF4, BAS1, and ACT2. The primers used for RT-PCR and qRT-PCR are listed in Table S1.
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The chemicals and reagents are as follows: IMDM cell medium, F12 cell medium (Gibco); osteogenic/adipogenic/chondrogenic induced differentiation medium (Suzhou Syagen); II-type collagenase, LPS, 3-MA, IGF-1Triton, and X-100 Tween (Sigma); Rabbit type II collagen primary antibody, rabbit Cleaved Caspase-3 primary antibody, rabbit Bax primary antibody, rabbit LC3 primary antibody, rabbit Beclin-1 primary antibody, rabbit β-actin primary antibody, rabbit p-Akt primary antibody, rabbit Akt primary antibody, rabbit p-mTOR primary antibody, rabbit mTOR primary antibody, HRP-labeled goat anti-rabbit secondary antibody, rabbit CD45 primary antibody, rabbit CD34 primary antibody, rabbit CD29 primary antibody, rabbit CD90 primary antibody, rabbit CD63 primary antibody, rabbit CD9 primary antibody, Rabbit GAPDH primary antibody, and rabbit TSG101 primary antibody (Abcam); TBS buffer, PBS buffer (Biosharp); Exosome Isolation Kit (Life technologies); primary antibody dilutions, secondary antibody dilutions, Trizol Lysate, ECL kit, PKH67 Staining Kit, BCA Protein Quantitative Kit, Annexin V-APC/PI Apoptosis Detection Kit, DAPI staining solution, RIPA Lysate (Medium), and PMSF (Beyotime, Jiangsu); 5 All-In-One RT MasterMix, SYBR Green Supermix (Abm), PAGE Gel Rapid Preparation Kit (12.5%) (Shanghai Epizyme); TNF-α ELISA Detection Kit, IL-1β ELISA Detection Kit, and Shanghai Enzyme Link.
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3

Investigating LEF-10 Aggregation and Late Gene Transcription

To investigate the effect of LEF-10 aggregation on late gene transcription, semi-quantitative RT-PCR was performed. Total RNAs were isolated with the TRIzol RNA extraction kit (Beijing CoWin Biotech) from 1×106Sf9 cells infected by vAcΔlef10/actinp-p10p:lef101-65-GFP or vAcΔlef10/actinp-p10p:lef10-GFP (Fig 1D) at 48 hpi. The RNA samples were then treated with RNase-Free DNaseI (Thermo). Reverse transcription was performed using the 5× All-In-One RT MasterMix (ABM), employing 2 μg total RNA as the template per reaction. The target DNA fragments were then amplified by PCR using the according primer pairs (listed in Table 1). After 25–29 cycles of amplification, the PCR samples were analyzed by 1% agarose gel electrophoresis.
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Total RNA was extracted from all samples according to instructions of the EASY Spin Plant RNA Rapid Extraction Kit (Aidlab Biotech, Beijing, China). The purity and concentration of all RNA samples were assessed using a Nanodrop 2000C spectrophotometer (Thermo Fisher Scientific, Wilmington, Delaware, DE, USA), and RNA quality was detected using 1 % agarose gels. Furthermore, cDNA was synthesized with 1 µg of total RNA using 5× All-In-One RT MasterMix (with an AccuRT Genomic DNA Removal Kit) (ABM, Vancouver, BC, Canada).
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Quantitative PCR (qPCR) was performed as previously described.50 (link) Total RNA was extracted using TRIzol reagent (Life Technologies, catalog number 15596-018). Equal amounts of total RNA from each sample were subjected to oligo (dT)-primed cDNA synthesis using 5× All-In-One RT MasterMix (Abm, catalog number G492). Reactions were run according to a standard protocol using SYBR Green Realtime MasterMix (TOYOBO, catalog number QPS-201) on a Roche Light Cycler 480 System (Roche Diagnostics). All results are presented in arbitrary units relative to human ACTIN mRNA expression. The specific primers used for qPCR are summarized in Supplementary Table S1.
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According to the manufacturer’s instructions, total RNA was extracted from bEnd.3 cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and miRNAs were isolated using a SanPrep Column microRNA Extraction Kit (Sangon Biotech, Shanghai, China). The 5× All-In-One RT MasterMix (abm, Richmond, BC, Canada) was used to reverse transcribe mRNA to cDNA. The cDNA and primers were mixed with 2X M5 HiPer SYBR Premix EsTaq (Mei5bio, Beijing, China) and then subjected to qRT–PCR. The levels of miRNAs were confirmed using a miRNA First Strand cDNA Synthesis (Tailing Reaction) Kit and a microRNA qPCR Kit (SYBR Green Method) (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. The 2−ΔΔCt method was used to calculate the expression levels of the mRNAs and miRNAs. The following primer sequences were used: 5′-TAACTATGACGGCTCGCTTAAC-3′ (forward) and 5′-ACAGCTACTTTGTTGTCCTTCT-3′ (reverse) for MCM2, 5′-CCGTAAAGACCTCTATGCCAAC-3′ (forward) and 5′- AGGAGCCAGAGCAGTAATCT-3′ (reverse) for Actb, which served as the internal control, and 5′-TAACAGTCTCCAGTCACGGCCA-3′ for mmu-miR-212-3p.
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TRIzol Reagent (Invitrogen) was used to extract total RNA from spleens or aortas. 100 ng/10 μL of spleen or aorta RNA were quantified with NanoDrop One (Thermo Scientific) and 5× All-In-One RT MasterMix (abm) was used for reverse transcription in Veriti 96-Well Thermal Cycler (Applied Biosystems). 7500 Real-Time PCR (Applied Biosystems) was used for qRT-PCR using specific primers (synthesized by Sangon Biotech) and Eva Green 2× qPCR Master Mix-Low ROX (abm). Reverse transcription and amplification conditions followed the reagent instructions. Data were analyzed via the 2-ΔΔCt method normalized to β-Actin. Sequences of primers were used as follows from 5′ to 3′ extremity:

β-Actin: GGCTGTATTCCCCTCCATCG (F); CCAGTTGGTAACAATGCCATGT (R);

Dnmt1: ATCCTGTGAAAGAGAACCCTGT (F);

CCGATGCGATAGGGCTCTG (R);

Dnmt3b: AGCGGGTATGAGGAGTGCAT (F);

GGGAGCATCCTTCGTGTCTG (R)

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Potato plant leaves were used when they reached 6 weeks old. A total of three leaves (from the top third to fifth compound leaf) from three separate plants were inoculated with P. infestans HB09-14-2. For each time point (0, 24, 48, 72 hours following P. infestans inoculation), three leaves were taken from individual plants and snap-frozen in liquid nitrogen. For plant defense hormone treatment, potato leaves were treated by spraying 1 mM ABA, 0.05 mM brassinolide (applied as epibrassinolide), 1 mM ethylene (applied as ACC), 1 mM salicylic acid, 1 mM methyl jasmonate and ddH2O. All solutions contained 1% dimethyl sulfoxide (DMSO). Six hours later three leaf disks (9 mm in diameter) were collected and frozen in liquid nitrogen. For RNA extraction we used the Plant Total RNA Kit (Zoman Biotechnologies, Beijing, China), and 5× All-In-One RT MasterMix (ABM) was used for the synthesis of the first-strand cDNA. Power SYBR Green was used for qRT–PCR experiments (Bio-Rad). The comparative ΔΔCt method was used for gene expression level analysis utilizing StEF1α as the reference gene for potato. Supplementary Data Table S1 lists the primer sequences.
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Human endometrial stromal cells were lysed with TRIzol reagent (Sigma, St. Louis, MO, USA) and total RNA was extracted in strict accordance with the instructions. Two micrograms of total RNA was added to a 20 μL system and reverse-transcribed into cDNA using 5 × All-In-One RT Master Mix (Abm, Canada). A SYBR Green PCR kit and MyiQ Single Colour Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) were employed for quantitative real-time PCR (qRT-PCR). The primer sequences used were as follows: KLF4, 5′-AGAGTTCCCATCTCAAGGCA-3′ and 5′-GTCAGTTCATCTGAGCGGG-3′; ATG5, 5′-AAAGATGTGCTTCGAGATGTGT-3′ and 5′-CACTTTGTCAGTTACCAACGTCA-3′; dPRL, 5′-CACTACATCCATAACCTCTC-3′ and 5′-ATGCTGACTATCAAGCTCAG-3′; IGFBP-1, 5′-TATGATGGCTCGAAGGCTCTC-3′ and 5′-GTAGACGCACCAGCAGAGTC-3′ and 18S rRNA, 5′-CGGCTACCACATCCAAGGAA-3′ and 5′-CTGGAATTACCGCGGCT-3′. The fold changes in the expression of each gene were measured by the 2−ΔΔCT method. The internal reference gene was 18S rRNA.
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Total RNA was isolated from anthers using TRIzol reagent (15596026, Invitrogen, Carlsbad, CA, USA). The concentration and purity of the isolated RNA were measured by a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher, Waltham, MA, USA). After removing genomic DNA with Dnase I (M6101, Promega, Madison, WI, USA), cDNA was synthesized using 5× All-In-One RT MasterMix (G592, abm, Vancouver, BC, Canada). The quantitative real-time PCR (qPCR) analysis was conducted with the corresponding primer set (Table S2) on a QuantStudio 5 Real-Time PCR system (ABI, Waltham, MA, USA), using TB Green Premix EX Tag (RR420A, Takara, Osaka, Japan); ZmActin1 was used as the internal control. Each sample had 3 biological replicates with 3 technical replicates, the amplification data were analyzed by the 2△△Ct method, and the quantitative results were given as means ± standard deviations.
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