Total RNA was extracted from the leaves of parsnip 6 h after mechanical wounding (Larbat et al., 2009 (link); Roselli et al., 2016 (link)) with the RNeasy plant extraction kit (Qiagen). The cDNA was generated using a High-Capacity RNA-to-cDNA system (Applied Biosystems) and random primers. The full-length cDNA sequence was then PCR-amplified using Platinum®Taq DNA Polymerase High Fidelity (Invitrogen) and primers with additional restriction sites and a 6xHis tag sequence at the 3′ end: CYP71AZ3for 5′-GGATCCATGGAGCCAGTATTTCTCTTTC-3′, CYP71AZ3rev 5′-GAATTCTTAGTGGTGATGGTGATGATGTGGAAACATATATTTTTTAGG-3′, CYP71AZ4for 5′-GGTACCATGGATCCAGCAGCTATC-3′, CYP71AZ4rev 5′-GAATTCTTAGTGGTGATGGTGATGATGTGGATGTACATATATTTTAGG-3′, CYP71AZ6 for 5′-ATGGATCCAGTAGTTATCTTTCTTGTCCTTGCTT-3′, and CYP71AZ6rev 5′-TTATGGACATATATATTTTTTAGGTCGAATGTAG-3′. PCR conditions were as follows: 2 min at 94°C, 35 cycles (30 s at 94°C, 30 s at 55°C, and 2 min at 68°C), and a final 10 min extension step at 68°C. PCR products were purified, cloned using a pCR®8/GW/TOPO® TA Cloning® Kit (Invitrogen), and sequenced. The resulting PCR products cloned in pCR®8/GW/TOPO® were sequenced and finally subcloned in the yeast expression vector pYeDP60-GW (Dueholm et al., 2015 (link)) using LR clonase II (Invitrogen).
Plant rneasy extraction kit
Manufactured by Qiagen
Sourced in United States, Germany
The Plant RNeasy extraction kit is a laboratory product designed for the isolation and purification of high-quality total RNA from plant samples. It provides a simple and efficient method for extracting RNA from a wide range of plant species and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Quantitect sybr green rt pcr kit, by Qiagen (2 mentions)
Reverse transcription kit, by Qiagen (2 mentions)
Dnase 1, by Qiagen (2 mentions)
Accupower 2x greenstar qpcr master mix, by Bioneer (2 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (2 mentions)
Pcr8 gw topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Lr clonase 2, by Thermo Fisher Scientific (1 mentions)
Platinum taq dna polymerase high fidelity, by Thermo Fisher Scientific (1 mentions)
Sybr green jumpstart taq readymix, by Merck Group (1 mentions)
Cfx96 real time system, by Merck Group (1 mentions)
Sybr green rt pcr kit, by Qiagen (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
Random primer dna labeling kit, by Takara Bio (1 mentions)
Hybond n nylon membrane, by Cytiva (1 mentions)
Hybond, by Cytiva (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Iq sybr green supermix kit, by Bio-Rad (1 mentions)
Cfx connect quantitative pcr machine, by Bio-Rad (1 mentions)
19 protocols using plant rneasy extraction kit
1
Cloning and Sequencing of Parsnip CYP71AZ Genes
2
Bee Homogenization and RNA Extraction
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Five bees from each sample were homogenized in bulk using standard methods (de Miranda et al., 2013) and total RNA was purified from the homogenate using the RNeasy plant extraction kit (Qiagen). The approximate RNA concentration of each sample was determined using the NanoDrop 2000 (Thermo Scientific), and the RNA samples were stored at -80 ºC until further use.
3
Quantifying Plant Gene Expression
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Total RNA (1 µg) were extracted from stems of 5-week-old wild-type and T3 homozygous transgenic lines using the Plant RNeasy extraction kit (Qiagen) and reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science). The cDNA preparations obtained were quality-controlled using tub8-specific oligonucleotides, and the pobA, ubiC and AtHCT transcripts were detected using specific oligonucleotides listed in Supplementary Table S6 . For the PCR, OneTaq (New England Biolabs) was used according to the manufacturer’s instructions, with 1 min of elongation time and 60 °C for the annealing temperature. Thirty (for pobA and ubiC expression) or 25 (for AtHCT expression) PCR cycles were used to amplify the DNA fragments.
4
RNA Extraction and Real-Time PCR Protocol
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Three samples of few inflorescences each were harvested for each genotype, and RNA was extracted with the Qiagen Plant RNeasy extraction kit. cDNA was synthetized with M-MMV reverse transcriptase (Invitrogen). Real-Time PCR was performed in technical quadruplicate using a Bio-Rad CFX96 Real-Time System and SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich). Primers used are listed in Table S2 .
5
RNA Extraction and Transcript Analysis
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Total RNA was isolated from the frozen samples using the Plant RNeasy extraction kit (Qiagen, Valencia, CA, USA). The splicing pattern of the intron-containing genes was analyzed by RT-PCR using the gene-specific primers listed in Additional file 8 as previously described [43 (link)]. Splicing efficiency was measured by real-time RT-PCR using the gene-specific primers listed in Additional file 9 as previously described [32 (link), 41 (link)]. The levels of chloroplast transcripts were measured by quantitative RT-PCR using the gene-specific primers listed in Additional file 10 . Real-time RT-PCR was carried out on a Rotor-Gene Q thermal cycler (Qiagen) using a SYBR Green RT-PCR kit (Qiagen). For northern blot analysis, four or five micrograms of total RNA were separated on a 1.2% formaldehyde-agarose gel and transferred to a Hybond-N+ nylon membrane (Amersham Biosciences, Parsippany, NJ, USA). The [α-32P]-labeled probes were synthesized using a random primer DNA labeling kit (TaKaRa Bio., Shiga, Japan). Hybridization, washing, and detection of signals were performed essentially as described previously [32 (link)].
6
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted with a Plant RNeasy extraction kit (Qiagen, Valencia, CA, USA). cDNA was synthesized with 3 μg of total RNA using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, Burlington, ON, Canada). RT-PCR proceeded for 30 cycles as follows: 94 °C, 15 s; 57 °C, 15 s; 72 °C, 1 min. qPCR was performed with a CFX Connect quantitative PCR machine (Bio-Rad, Hercules, CA, USA). The iQ SYBR Green Supermix kit (Bio-Rad) was used for qPCR analysis. The reaction primers used in RT-PCR or qPCR are listed in Table S1 .
7
Quantitative RT-PCR Analysis of Leaf Transcripts
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Total RNA was isolated from several leaves using the Plant RNeasy extraction kit (Qiagen, Hilden, Germany). cDNA was synthesized from 1 μg of RNA (DNase treated) using iScript™ cDNA Synthesis Kit (BioRad, Hercules, CA, USA). All reactions were done in triplicate on three biological replicates. The following oligonucleotides were used: STY8QRT-PCR for 5‘-CCACGGATGGAACTGATGAGT-3‘, STY8QRT-PCR rev 5′-TACACGATCAGGCTTGAGAAA-3′, STY17QRT-PCR for 5′-AAGGTTTAAAAGATGCATTGA-3′, STY17QRT-PCR rev 5′-CATCAGTTCCATCCGTAGGTA-3′, STY46QRT-PCR for 5′- AGGTGCCAGAACGCATGTTCC-3′, STY46QRT-PCR rev 5′-TTGATAGCAACTTCCTGGCTA-3′, RCE1_qpcr_fr 5′-CTGTTCACGGAACCCAATTC-3′, RCE1_qpcr_rev 5′-GGAAAAAGGTCTGACCGACA-3′. The relative abundance of all transcripts amplified was normalized to RCE1 (At4g36800). For quantitative RT-PCR, the SYBR Green real-time PCR master mix (Roche, Basel, Switzerland) was used, and the reaction was performed in a Bio-Rad CFX96 real-time PCR detection system. Forty-five cycles were performed as follows: 1 s at 95 °C, 7 s at 49 °C, 19 s at 72 °C, and 5 s at 79 °C.
8
Real-time PCR Transcription Analysis
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Total RNA was extracted from samples using a plant RNeasy extraction kit (Qiagen, Valencia, CA, USA). Transcription analyses of genes were conducted using real-time PCR with a Rotor-Gene Q real-time thermal cycling system (Qiagen, CA, USA), QuantiTect SYBR Green RT-PCR kits (Qiagen, CA, USA), 200 ng RNA samples, 35 cycles and the primers listed in Additional file 2 : Table S1 as described previously [65 (link), 66 (link)].
9
Quantification of Target Gene Expression
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To determine the expression levels of target genes, total RNA was extracted from the frozen tissues using the Plant RNeasy extraction kit (Qiagen, http://www.qiagen.com ), and 200ng of RNA was reverse transcribed and amplified using the One-step RT-PCR kit (Qiagen) with the gene-specific primers listed in Supplementary Table S1 . To detect 21-mer mature amiRNAs in the transgenic plants, 20 μg of total RNA was separated via denaturing 12% PAGE and transferred to a nylon membrane, as previously described (Kim et al., 2010 (link); Jung and Kang, 2014 (link)). RNA blots were hybridized with a radiolabeled probe complementary to the amiRNA, and the signals were detected using a FLA7000 Phosphorimager (GE Healthcare Life Sciences, http://www.gelifesciences.com ).
10
Total RNA Extraction and Quantification
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Total RNA (1 μg) was extracted from stems of 5-week-old wild-type and T3 homozygous transgenic lines using the Plant RNeasy extraction kit (Qiagen, Valencia, CA, USA) and reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Indianapolis, IN, USA). The cDNA preparations obtained were quality-controlled using the tub8-specific oligonucleotides Tub8-fw and Tub8-rv (Table S1 in Supplementary Material), and the AdoMetase transcripts were detected using the oligonucleotides AdoMetase-fw and AdoMetase-rv (Table S1 in Supplementary Material).
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