To detect mRNA expression of 11 PcNLP targeted genes in the silenced lines, gene-specific primers of each PcNLP gene were designed; these are listed in Additional file 4 : Table S1. The β-Actin, β-Tubulin, and Ubc (ubiquitin-conjugating enzyme) of P. capsici[69 (link)] were used as constitutively expressed endogenous controls and were used jointly as a reference to relate to the microarray data of the qRT-PCR detection. Each transformed line was first grown in 10% V8-juice liquid medium for three days at 25°C, and then total RNA was extracted from freeze-dried filtered mycelium based on the TRIZOL procedure (Invitrogen). Total RNA extractions of the different silenced lines and qRT-PCR were done as described above. WT is wild strain SD33; CK transformation is a strain expressing only the selected gene. SYBR green qRT-PCR assays were performed to determine individual PcNLP gene expression at the transcriptional levels. The expression levels of individual genes in SD33 or CK were assigned the value of 1.0 to allow comparison between lines. The threshold cycle (CT) values were determined automatically by instrument, and the fold changes of each gene were calculated by the equation 2-△△CT according to a previous description [70 (link)]. Results were obtained from three repeated trials.
Trizol procedure
Manufactured by Thermo Fisher Scientific
Sourced in United States
TRIZOL is a procedure for the isolation of total RNA from various biological samples. It is a single-step method that uses a phenol-chloroform extraction to separate RNA from DNA and proteins.
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Lab products found in correlation
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Qiaamp dna mini extraction kit, by Qiagen (1 mentions)
Abi 7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Primescrip rt reagent kit, by Takara Bio (1 mentions)
Sybr premix ex taqtm, by Takara Bio (1 mentions)
Humanht 12 v4 expression beadchip, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Rnalater, by Merck Group (1 mentions)
Polytron pt10 35, by Kinematica (1 mentions)
Quantitect sybr green pcr kit, by Qiagen (1 mentions)
Purelink pcr micro kit, by Thermo Fisher Scientific (1 mentions)
Solid 4 slide, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Dnase treatment, by Qiagen (1 mentions)
Nanodrop spectrophotometer, by Avantor (1 mentions)
Qiazol reagent, by Qiagen (1 mentions)
Hs01060665 g1, by Thermo Fisher Scientific (1 mentions)
Superscript 2 retrotranscriptase, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
14 protocols using trizol procedure
1
qRT-PCR Analysis of PcNLP Genes
2
Genetic Analysis of SPG4, SPG3A, and SPG31 in Taiwanese
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Genomic DNA was extracted from peripheral blood leukocytes for genetic analysis, and mutation screenings were conducted in the following order. First, nucleotide substitutions and small deletions or insertions in all exons and their adjacent splicing sites of the SPG4 gene (SPAST) were analyzed by polymerase chain reactions and direct sequencing. If a mutation was not detected, multiplex ligation-dependent probe amplification (MLPA) was performed to detect exonic deletions in SPAST. Finally, for the cases with no detected mutations in SPAST, sequence analysis and MLPA of the SPG3A gene (ATL1) and sequence analysis of the SPG31 gene (REEP1) were performed. Detected mutations were checked in 100 control DNA samples collected from unrelated ethnic Han Taiwanese.
MLPA was performed using SALSA MLPA kits (P165-B1 HSP probemix; MRC-Holland, Amsterdam, Netherlands) according to the manufacturer’s instructions. For each sample, a normalized value ratio for a relative peak area between 0.8 and 1.2 was considered normal. A heterozygous deletion was expected with a ratio between 0.3 and 0.7, and a heterozygous duplication between 1.3 and 1.7. Total mRNA from the blood leukocytes was extracted using Trizol procedure (Invitrogen, Carlsbad, CA, United States). mRNA analysis was performed as described previously [12 (link)] to assess alternations in gene transcription due to SPAST mutations.
MLPA was performed using SALSA MLPA kits (P165-B1 HSP probemix; MRC-Holland, Amsterdam, Netherlands) according to the manufacturer’s instructions. For each sample, a normalized value ratio for a relative peak area between 0.8 and 1.2 was considered normal. A heterozygous deletion was expected with a ratio between 0.3 and 0.7, and a heterozygous duplication between 1.3 and 1.7. Total mRNA from the blood leukocytes was extracted using Trizol procedure (Invitrogen, Carlsbad, CA, United States). mRNA analysis was performed as described previously [12 (link)] to assess alternations in gene transcription due to SPAST mutations.
3
Isolation of Trabecular Bone RNA and DNA
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Tibial plateaus were obtained in collaboration with the Department of Orthopedic Surgery (Hospital Lariboisiere) from patients undergoing knee replacement surgery due to osteoarthritis. This procedure has been approved by the local Ethics Committee (#06NICB of CCP Ile de France IV). For polymorphism studies, the patients were only females (n = 31) between 52 and 86 years old and were processed as described previously.38 Briefly, trabecular bone “carrot” samples were obtained from the inner part of the tibial plateau using a 2‐mm diameter biopsy trocar, and frozen in liquid nitrogen. Only tibial plateaus containing trabecular bone of apparent good quality and remote from the areas affected by osteoarthritis were selected. After crushing the trabecular samples in liquid nitrogen using a mortar and a pestle, total RNA was extracted using the TRIzol™ procedure (Invitrogen). Genomic DNA was extracted following the same crushing procedure, but using a QIAamp DNA Mini extraction kit from Qiagen.
4
Quantifying hnRNP-L Expression in Bladder Cancer
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Total RNAs was isolated from cultured cell lines or human bladder cancer specimens using Trizol procedure (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions. CDNAs were synthesized utilizing the PrimeScripTM RT reagent Kit (Takara) from 1μg of total RNA in 20 μl of reaction volume. Reaction mixture for qPCR executed on the ABI 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, CA, USA) by using SYBR Premix Ex TaqTM (Takara Clontech, Kyoto, Japan). GAPDH fragment was quantified as an internal control. The primer sequences were: Human hnRNP-L: 5′-TTGTGGCCCTGTCCAGAGAATT-3′ (forward) and 5′-GTTTGTGTAGTCCCAAGTATCCTG-3′ (reverse); human GAPDH: 5-ACAGTCAGCCGCATCTTCTT-3′ (forward) and 5′-GACAAGCTTCCCGTTCTCAG-3′ (reverse). Three independent replicates were executed for each experiment. The relative levels of each sample in comparison to control GAPDH was derived using the 2-ΔΔCt method.
5
Gene Expression Profiling of Gal-7 in Cells and Tumors
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RNA was extracted from tissue culture cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. A total of 500 ng of RNA from every sample was labeled and hybridized in the HumanHT-12v4 expression BeadChip (Illumina) following standard procedures of the Genomic Core facility at the DKFZ. For the tumors, fresh-frozen xenotransplants isolated from nude mice were homogenized in a Precellys® 24 tissue homogenizer (Bertin Tech. USA). Subsequently, RNA from tumors was isolated using standard TRIZOL procedure (Invitrogen) and Direct-zol RNA (Zymo Research) following the manufacturer’s instructions. Samples within the groups were pooled. A total of 500 ng of RNA from every pooled group was labeled and hybridized in the HumanHT-12v4 expression BeadChip (for human genes) or the mouseWG-6v2 expression BeadChip (for mouse genes) following standard procedures of the Genomic Core facility at the DKFZ. Bioinformatic data analysis was performed using Chipster [18 (link)] software version 3.1. mRNAs with a fold change of at least 2 between Gal-7 negative and Gal-7 positive cells or tumors were considered significant and were used for further analysis.
6
RNA Isolation from Jejunal Samples
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Jejunal samples were stored in RNAlater (Sigma, USA) and RNA was isolated using the TRIzol procedure (Invitrogen, USA). Tissues were homogenised with a Polytron PT10-35 (Kinematica, Switzerland) at medium speed for 10 s on ice. RNA was dissolved in sterile water and precipitated with sodium acetate. Quality and concentration were measured on a NanoDrop 1000 Spectrophotometer (Thermo Scientific, USA).
7
Northern Blot Analysis of Gene Expression
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For Northern blot analyses, strains were grown on CM overlayed with Cellophane for at least 3 days. The mycelium was harvested, lyophilized and the RNA was isolated via the Trizol procedure (Invitrogen, Groningen, The Netherlands). Samples of 20 μg were loaded to an agarose gel and transferred to Hybond-N+ membranes. Northern blot hybridizations were done according to the method of Church and Gilbert (1984) (link). The different probes used for expression analysis were generated by PCR with the primer pairs 40/41 (Hac1) and 42/43 (BiP/Kar2).
8
Fungal and Plant Gene Expression
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Total RNA was isolated from freeze-dried, fungal material collected from the stated liquid culture or from fungal infected leaf tissues, was prepared using the TRIZOL procedure (Invitrogen). Total RNA was used for all RT-PCR and Real-Time RT-PCR analyses. First-strand cDNA was synthesised from total RNA using the SuperScript III First_Strand Synthesis System for RT-PCR (Invitrogen). A 5 μg aliquot of total RNA primed with oligo(dT)20 was used in a 20 μl reaction, following the suppliers instructions. The resulting cDNA was analysed by Real-Time RT-PCR using a QuantiTect SYBR Green PCR Kit (Qiagen), following the supplier’s instruction. A 0.5 μl aliquot of cDNA was used in each 20 μl PCR reaction, with an annealing temperature of 60°C. Primers were used at a final concentration of 0.25 μM. Real-Time RT-PCR reactions were run for 40 cycles and analysed using an ABI 7500 Real Time PCR System. The relative expression of each fungal or plant gene was determined by normalisation with the constitutively expressed Z. tritici beta-tubulin gene [48 (link)] or with the TaCdc48 gene for T. aestivum [31 (link)]. All oligonucleotides used are listed in S5 Table .
9
Hippocampal SAGE Sequencing in Aged and Young Mice
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Total RNA was extracted from one hippocampus from each animal using the TRIzol procedure (Invitrogen) and processed in parallel. RNA quality was assessed using an Agilent Bioanalyser 2100 and quantified using a Qubit 2.0 Fluorometer (Invitrogen). All RNA samples had an RNA Integrity Number score greater than 8, with an average of 8.43. SAGE tag library preparation was performed according to the SOLiD SAGE kit with Barcoding Adaptor Module (Invitrogen). Approximately 3 μg of RNA was used to isolate polyadenylated RNA using poly-dT Dynabeads and converted to cDNA using Superscript III Reverse Transcriptase. Samples were digested with sequence-specific Endonuclease Nla III restriction enzyme and ligated to barcoded adaptor A. The fragments were then released from the Oligo (dT) EcoP magnetic beads using EcoP15I restriction enzyme. The resulting fragments were then ligated to adapter B which contains the P1 sequence. 130 bp ligation products were PCR-amplified and purified using PureLink PCR Micro Kit (Invitrogen). Barcoded libraries were then quantified using a Qubit Fluorometer and pooled in equimolar volumes before emulsion PCR and sequenced on one full SOLiD4 slide (Applied Biosystems). From four groups belonging to the same MWM cohort–aged EE, aged SH, young EE, and young SH–three samples from each group were run in parallel as biological replicates.
10
Viral Metagenomics from Biopsies
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Total RNA was extracted from biopsies using the Trizol procedure (Thermo Fischer Scientific), purified on RNeasy column including a DNase treatment (Qiagen), and randomly amplified [32 (link)] for each patient individually. High-molecular-weight DNA was used for library construction and high-throughput sequencing (HTS) was subcontracted to DNA Vision (Charleroi, Belgium) on a HiSeq 2000 instrument, generating an average of 92 million reads per sample (min = 46 M; max = 162 M). Sequences were trimmed and then screened for viral sequences using the protein version of RVDB database [33 (link)]. Additional screening for bacteria and other non-viral microorganism sequences was performed using Centrifuge [34 (link)] and Metaphlan2 [35 (link)]. The reads and contigs of interest were mapped using the Geneious mapper of Geneious 11.1.5 [36 (link)] with the medium sensitivity/fast parameters.
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