Iscript advanced reverse transcriptase, by Bio-Rad - Product details

1

Real-time qPCR Assay Protocol

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Real-time (RT) quantitative polymerase chain reaction (qPCR) was run
using BioRad Custom PrimePCR plates, as per the manufacturer’s
instructions. Briefly, cDNA was synthesized using 5x iScript Advanced Reaction
Mix and iScript Advanced Reverse Transcriptase (BioRad) at 46 °C for 20
min then 95 °C for 1 min. cDNA samples were mixed with 2x iTaq Universal
SYBR-green Supermix and iScript Reverse Transcriptase. The RT qPCR was performed
at 50 °C for 10 min, 95 °C for 1 min, 95 °C for 15 sec (40
cycles), 60 °C for 60 sec (40 cycles). Data was analyzed using CFX
manager and presented as 2^-ΔCt.

Lloyd A.F., Davies C.L., Holloway R.K., Labrak Y., Ireland G., Carradori D., Dillenburg A., Borger E., Soong D., Richardson J.C., Kuhlmann T., Williams A., Pollard J.W., des Rieux A., Priller J, & Miron V.E. (2019). Central nervous system regeneration is driven by microglia necroptosis and repopulation. Nature neuroscience, 22(7), 1046-1052.

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2

High-Throughput Transcriptome Profiling via mmPCR–seq

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The mmPCR–seq was performed as described in Zhang et al. (2014) (link). Briefly, total RNA is extracted from cells using a QIAGEN Micro or Mini RNeasy kit (QIAGEN cat:74004 or 74104) and reverse transcribed using iScript Advanced reverse transcriptase (Bio-Rad). The cDNAs were purified using Ampure XP Beads (Beckman Coulter), with an elution volume of 10 μL. 250 ng of cDNA was used in a preamplification reaction and amplified cDNA was purified using Ampure XP Beads and eluted in 10 μL. 50 ng of pre-amplified cDNA was loaded into each well of an Access Array microfluidic chip (Fluidigm). The PCR reactions were performed on the Access Array System (Fluidigm) using KAPA2G 5X Fast Multiplex PCR Mix (Kapa Biosystems). Barcodes were added in a second round of PCR using Phusion DNA polymerase (NEB cat: M0531S). Samples were sequenced with 76 base-pair paired-end reads using an Illumina NextSeq (Illumina, San Diego, CA).

Freund E.C., Sapiro A.L., Li Q., Linder S., Moresco J.J., Yates JR I.I.I, & Li J.B. (2020). Unbiased Identification of trans Regulators of ADAR and A-to-I RNA Editing. Cell reports, 31(7), 107656.

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3

Multiplexed qRT-PCR Sequencing Protocol

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We have previously described our mmPCR–seq method in detail^{19 (link)}. In brief, RNAs were reverse transcribed using either SuperScript III (Invitrogen) or iScript advanced reverse transcriptase (Bio-Rad). The cDNAs were purified using the MinElute PCR Purification Kit (Qiagen), with an elution volume of 15 μl or less. For brain samples, at least 200 ng cDNA was loaded into each well of an Access Array microfluidic chip (Fluidigm). For non-brain samples, at least 400 ng cDNA was loaded. The PCR reactions were performed on the Access Array System (Fluidigm) using 5× KAPA2G Multiplex PCR Mix (Kapa Biosystems). The primer sequences for both human and mouse are provided in Supplementary File 2. Barcodes were added in a second round of PCR using Phusion DNA polymerase (Finnzymes). Samples were sequenced on HiSeq 2000 (Illumina) to produce paired 101-bp reads. Details of all the samples are provided in Supplementary File 8.

Tan M.H., Li Q., Shanmugam R., Piskol R., Kohler J., Young A.N., Liu K.I., Zhang R., Ramaswami G., Ariyoshi K., Gupte A., Keegan L.P., George C.X., Ramu A., Huang N., Pollina E.A., Leeman D.S., Rustighi A., Sharon Goh Y.P., Chawla A., Del Sal G., Peltz G., Brunet A., Conrad D.F., Samuel C.E., O’Connell M.A., Walkley C.R., Nishikura K, & Li J.B. (2017). Dynamic landscape and regulation of RNA editing in mammals. Nature, 550(7675), 249-254.

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4

Efficient RNA Extraction and cDNA Synthesis

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Cell pellets from the different treatments were initially lysed with 1 ml of TRIzol reagent. Chloroform (0.2 ml) was added, vortexed, incubated at 15–30°C for 2–3 min, and centrifuged at 10,000 g for 15 min at 2–8°C. The aqueous phase of the lysed samples was transferred to a fresh tube, and the RNA was precipitated by mixing with isopropyl alcohol (0.5 ml). After centrifugation, 75% ethanol was used to wash the RNA pellet. After centrifugation at 7,500 g for 5 min at 2–8°C, the pellet was left to dry for 15 min and then dissolved in water (RNase free). The purity and RNA concentration were measured using a NanoDrop (Thermo Fischer Scientific). Using iScript advanced reverse transcriptase from Bio-Rad, the cDNA strands were synthesized based on the mRNA. The solutions were loaded into 0.2 ml tubes and included 5X iScript advanced reaction mix (4 µl) (containing primers), reverse transcriptase (١ µl), sample (١.٥ µg/٧.٥ µl), and water (٧.٥ µl) in a total of ٢٠ µl. The thermal cycling for reverse transcription steps included ٣٠ min at 42°C and 5 min at 85°C.

Mendonca P., Taka E, & Soliman K.F. (2019). Proteomic analysis of the effect of the polyphenol pentagalloyl glucose on proteins involved in neurodegenerative diseases in activated BV-2 microglial cells. Molecular Medicine Reports, 20(2), 1736-1746.

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5

Reverse Transcription Primer-Based cDNA Synthesis

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The cDNA strand was synthesized from the mRNA using iScript advanced reverse transcriptase from Bio-Rad. A solution of 4µl of the 5X iScript advanced reaction mix (containing primers), 1µl of reverse transcriptase, 5µl of the sample (250ng/5µl) for RT-PCR assay and 7.5µl of the sample (1.5µg/7.5µl) for RT-PCR with individual primers, and water were added to 0.2ml tubes, in a total of 20µl. The Reverse Transcription thermal cycling program included two steps: 42^oC for 30 min and then 85^oC for 5 min.

Mendonca P., Taka E., Bauer D., Reams R.R, & Soliman K.F. (2018). The Attenuating Effects of 1,2,3,4,6 Penta-O-Galloyl-β-D-Glucose on Pro-Inflammatory Responses of LPS/IFNγ-activated BV-2 Microglial Cells through NFƙB and MAPK Signaling Pathways. Journal of neuroimmunology, 324, 43-53.

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Comprehensive Retrotransposon Expression Analysis

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RNA was isolated using Trizol following manufacturer’s instruction (Life Technologies, Cat. # 15596). cDNA was reverse-transcribed using iScript Advanced Reverse-Transcriptase (Bio-Rad, Cat. # 1725037). All real-time qPCR analyses were performed using SYBR FAST qPCR Master Mix (Kapa Biosystems, Cat. # KK4604), following manufacturer’s protocol. Real time PCR analyses on retrotransposons detect their expression at the family level, using primers designed from the corresponding consensus sequences. Actin was used as a reference for both mRNA and retrotransposon quantitation in real time PCR analyses. All real time PCR primers used in our studies are listed in Table S5.

Kinisu M., Choi Y.J., Cattoglio C., Liu K., de Bezieux H.R., Valbuena R., Pum N., Dudoit S., Huang H., Xuan Z., Kim S.Y, & He L. (2021). Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes. Cell reports, 37(6), 109982.

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7

cDNA Synthesis and RT-PCR Amplification

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The cDNA strands were synthesized from the mRNA using iScript advanced reverse transcriptase from Bio-Rad. A solution of 4 μl of the 5X iScript advanced reaction mix (containing primers), 1 μl of reverse transcriptase, 7.5 μl of the sample (1.5 μg/reaction), and 7.5 μl of water was combined in a 0.2 ml tubes, in a total volume of 20 μl. The thermal cycling program for the reverse transcription included two steps: 46°C for 20 min and then 95°C for 1 min. RT-PCR amplification was performed following the manufacturer protocol (Bio-Rad). A 1 μl of the sample (200 ng cDNA/reaction), 10μl of the master mix, 1 μl of primer, and 8 μl of water were combined into each well. The thermal cycling process included an initial hold step at 95°C for 2 min and denaturation at 95°C for 10 sec, followed by 39 cycles of 60°C for 30 sec (annealing/extension), and 65°C—95°C for 5 sec/step (melting curve) using the Bio-Rad CFX96 Real-Time System (Hercules, CA, USA). The selected primers were specific to each gene of interest. The UniqueAssay ID for CCL2/MCP1 primer was qHsaCID0011608, and the for IKBKE primer was qHsaCID0014831.

Mendonca P., Horton A., Bauer D., Messeha S, & Soliman K.F. (2019). The inhibitory effects of butein on cell proliferation and TNF-α-induced CCL2 release in racially different triple negative breast cancer cells. PLoS ONE, 14(10), e0215269.

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8

Single-Cell Analysis of MERVL Expression

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RNA was isolated by Trizol extraction following manufacturer’s instruction (Life Technologies, Cat. # 15596). cDNA was reverse-transcribed using iScript Advanced Reverse-Transcriptase (Bio-Rad, Cat. # 1725037). For single colony analysis, cDNA was prepared using a Single Cell-to-Ct qRT-PCR kit (Life Technologies, Cat. # 4458236). All real-time qPCR analyses were performed using SYBR FAST qPCR Master Mix (Kapa Biosystems, Cat. # KK4604). All primers used are listed in Table S6. To detect MERVL expression, four pairs of primers were designed to amplify specific regions of MERVL (Fig. 3C) and yielded similar results (data now shown). One pair of primers detecting the MERVL pol region was used for all other MERVL real-time PCR analyses.

Choi Y.J., Lin C.P., Risso D., Chen S., Tan M.H., Li J.B., Wu Y., Chen C., Xuan Z., Macfarlan T., Peng W., Kim S.Y., Speed T.P, & He L. (2017). Deficiency of microRNA miR-34a expands cell fate potential in pluripotent stem cells. Science (New York, N.Y.), 355(6325), eaag1927.

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9

Comprehensive Retrotransposon Expression Analysis

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RNA was isolated using Trizol following manufacturer’s instruction (Life Technologies, Cat. # 15596). cDNA was reverse-transcribed using iScript Advanced Reverse-Transcriptase (Bio-Rad, Cat. # 1725037). All real-time qPCR analyses were performed using SYBR FAST qPCR Master Mix (Kapa Biosystems, Cat. # KK4604), following manufacturer’s protocol. Real time PCR analyses on retrotransposons detect their expression at the family level, using primers designed from the corresponding consensus sequences. Actin was used as a reference for both mRNA and retrotransposon quantitation in real time PCR analyses. All real time PCR primers used in our studies are listed in Table S5.

Kinisu M., Choi Y.J., Cattoglio C., Liu K., de Bezieux H.R., Valbuena R., Pum N., Dudoit S., Huang H., Xuan Z., Kim S.Y, & He L. (2021). Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes. Cell reports, 37(6), 109982.

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10

Real-time qPCR Assay Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols

Real-time (RT) quantitative polymerase chain reaction (qPCR) was run
using BioRad Custom PrimePCR plates, as per the manufacturer’s
instructions. Briefly, cDNA was synthesized using 5x iScript Advanced Reaction
Mix and iScript Advanced Reverse Transcriptase (BioRad) at 46 °C for 20
min then 95 °C for 1 min. cDNA samples were mixed with 2x iTaq Universal
SYBR-green Supermix and iScript Reverse Transcriptase. The RT qPCR was performed
at 50 °C for 10 min, 95 °C for 1 min, 95 °C for 15 sec (40
cycles), 60 °C for 60 sec (40 cycles). Data was analyzed using CFX
manager and presented as 2^-ΔCt.

Lloyd A.F., Davies C.L., Holloway R.K., Labrak Y., Ireland G., Carradori D., Dillenburg A., Borger E., Soong D., Richardson J.C., Kuhlmann T., Williams A., Pollard J.W., des Rieux A., Priller J, & Miron V.E. (2019). Central nervous system regeneration is driven by microglia necroptosis and repopulation. Nature neuroscience, 22(7), 1046-1052.

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Iscript advanced reverse transcriptase

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16 protocols using iscript advanced reverse transcriptase

Real-time qPCR Assay Protocol

High-Throughput Transcriptome Profiling via mmPCR–seq

Multiplexed qRT-PCR Sequencing Protocol

Efficient RNA Extraction and cDNA Synthesis

Reverse Transcription Primer-Based cDNA Synthesis

Comprehensive Retrotransposon Expression Analysis

cDNA Synthesis and RT-PCR Amplification

Single-Cell Analysis of MERVL Expression

Comprehensive Retrotransposon Expression Analysis

Real-time qPCR Assay Protocol

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