Xiu Yun Lei1, Ming Ye Zhang1, Yan Jun Ma1, Jian Wen Wang1*

Transcriptomic responses involved in enhanced production of hypocrellin A by addition of Triton X-100 in submerged cultures of Shiraia bambusicola

Xiu Yun Lei1, Ming Ye Zhang1, Yan Jun Ma1, Jian Wen Wang1*

* * JW Wang

;

1College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China

Supporting Information

Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Report

Information designed according to the MIQE guidelines as essential (E) is presented Desirable information (D) is attached if it is available

EXPERIMENTAL DESIGN

Definition of experimental and control groups (E)

Control group: The seed culture (10%, v/v) of S. bambusicola S8 was poured into a 250-mL Erlenmeyer flask containing 100 mL of the basic liquid medium containing (per liter): 200 g potato, 20 g glucose, 1 g KH2PO4, 0.5 g MgSO4, 0.5 g KCl, 0.01 g FeSO4·7H2O, 3 g yeast extract, 10 g peptone, and pH 6.5. The cultures were maintained on a rotary shaker at 200 rpm and at 28°C for an overall culture period of 8 d.

Experimental group: Triton X-100 was added at 2.5% (w/v) at 36 h of initial cultures. The cultures were maintained on a rotary shaker at 200 rpm and at 28°C for an overall culture period of 8 d.

Number within each group (E)

Each experiment was carried out in triplicate.

Assay carried out by core lab or investigator's lab? (D)

Assay was carried out by investigator’s lab.

SAMPLE

Description (E)

The samples were collected at the end of each experiment.

Volume/mass of sample processed (D)

The sample volume was 100 mL.

Microdissection or macrodissection (E)

Not applied.

Processing procedure (E)

The mycelia were filtered through the filter cloth and the medium was discarded. The mycelia were then washed three times with distilled water. Samples were stored at -80 °C until use.

If frozen - how and how quickly? (E)

Mycelia were immediately placed into an ultra-low temperature freezer (-80 °C).

If fixed, with what and how quickly? (E)

Not applied.

Sample storage conditions and duration (especially for FFPE samples) (E)

Samples were stored at -80 °C till RNA extraction (maximally for one week).

NUCLEIC ACID EXTRACTION

Procedure and/or instrumentation (E)

Total RNA was extracted using RNAprep pure Plant Kit (Tiangen, Beijing, China) following the manufacturer’s instructions.

Name of kit and details of any modifications (E)

RNAprep pure Plant Kit (Tiangen, Beijing, China)

Details of DNase or RNAse treatment (E)

RNAprep pure Plant Kit (Tiangen, Beijing, China) was used following the manufacturer’s instructions.

Contamination assessment (DNA or RNA) (E)

A level of DNA contamination was determined by qPCRs with treated RNA and corresponding cDNA samples. Cq-values were compared - only samples without signals in the reaction with RNA or with a higher difference than 15 Cqs between RNA and cDNA samples were assessed to be free of DNA contamination

Nucleic acid quantification (E)

RNA concentration was quantified spectrophotometrically using Quawell Q5000 UV-Vis Spectrophotometer (Quawell, San Jose, CA,USA).

Instrument and method (E)

Quawell Q5000 UV-Vis Spectrophotometer (Quawell, San Jose, CA,USA) was used according to manufacturer’s protocol.

Purity (A260/A280) (D)

The ratio A260/A280 was between 1.8 and 2.1 indicating good purity in each sample.

Yield (D)

Approximately 25-54 μg RNA was extracted from each sample.

RNA integrity method/instrument (E)

RNA integrity was verified by gel electrophoresis.

RIN/RQI or Cq of 3' and 5' transcripts (E)

The RIN value of each sample was about 10.

Inhibition testing (Cq dilutions, spike or other) (E)

Not determined

REVERSE TRANSCRIPTION

Complete reaction conditions

The first cDNA strand was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, San Jose, USA) according to the manufacturer’s instructions. In a sterile microcentrifuge tube, was added 2 µL RNA, 1 µL 1.5 µg/µL Oligo (dT)18 primer and 9 µL nuclease-free H2O. The tube was heated at 65°C for 5 min and then was placed in ice. Subsequently, the tube was added 4 µL 5 × reaction buffer, 2 µL 10 mM dNTPs, 1 µL reverse transcriptase and 1 µL ribolock RNase inhibitor. The tube was incubated at 45°C for 1 h and heated at 70°C for 5 min.

Amount of RNA and reaction volume (E)

2 µL RNA (250 µg/µL), 20 μl reaction volume

Priming oligonucleotide (if using GSP) and concentration (E)

Oligo (dT)18, 1.5 µg/µL

Reverse transcriptase and concentration (E)

RevertAid™ Reverse Transcriptase, 200 U/µL

Temperature and time (E)

65°C for 5 min, 45°C for 1 h and 70°C for 5 min

Cqs with and without reverse transcription (D)

Cqs with reverse transcription < 40; Cqs without reverse transcription - undetected or difference higher than 15 Cqs between samples with and without reverse transcription

Storage conditions of cDNA (D)

Stored at -20 °C

qPCR TARGET INFORMATION

Gene symbol (E)

Reference gene: 18S (18S ribosomal RNA)

Polyketide synthase genes: comp11506_c0_seq1, comp21519_c0_seq1

Monooxygenase gene: comp14060_c0_seq1

O-methyltransferase gene: comp16507_c0_seq1

MFS transporter gene: comp13844_c0_seq1

ABC transporter gene: comp2509_c0_seq1

Antioxidant enzyme genes: comp15524_c0_seq1, comp5716_c0_seq2, comp13823_c0_seq1, comp14382_c0_seq1

ROS biosynthesis gene: comp2367_c0_seq1

Fatty acid desaturase genes: comp12623_c0_seq4, comp13666_c0_seq1, comp6499_c0_seq4, comp13878_c0_seq1

Sequence accession number (E)

Not applied.

Amplicon length (E)

18S 103 bp, comp11506_c0_seq1 257 bp, comp21519_c0_seq1 119 bp, comp14060_c0_seq1 177 bp, comp16507_c0_seq1 156 bp, comp13844_c0_seq1 207 bp, comp2509_c0_seq1 254 bp, comp15524_c0_seq1 231 bp, comp5716_c0_seq2 260 bp, comp13823_c0_seq1 149 bp, comp14382_c0_seq1 124 bp, comp2367_c0_seq1 189 bp, comp12623_c0_seq4 235 bp, comp13666_c0_seq1 129 bp, comp6499_c0_seq4 176 bp, comp13878_c0_seq1 202 bp.

In silico specificity screen (BLAST, etc) (E)

Not applied

Location of each primer by exon or intron (if applicable) (E)

Exon

What splice variants are targeted? (E)

Not applied

qPCR OLIGONUCLEOTIDES

Primer sequences (E)

Genes / Name of primer / Sequence (5’-3’)
18S / 18S-F
18S-R / ACGCAGCGAAATGCGATAAG
CAAATTGTGCTGCGCTCCAA
comp11506_c0_seq1 / 11506-F
11506-R / GCAGATACGCCCCTCACTAC
GTCGCTGGTAATATCGCCCA
comp21519_c0_seq1 / 21519-F
21519-R / GGAAGCGTTTGTCACGCAAT
TCTTGAGGTGCCGACAAGAC
comp14060_c0_seq1 / 14060-F
14060-R / TCGGAAGCCTCGTTCGAAAA
GTGAGAGGCCTCCTTTTCCC
comp16507_c0_seq1 / 16507-F
16507-R / ATCATGATGCGCGATGGAGT
AGATGATTGCACAAGGCCGA
comp13844_c0_seq1 / 13844-F
13844-R / CTTGGCCAGCCAAAAAGACC
GCGCAGCCATGAGTGTAATG
comp2509_c0_seq1 / 5429-F
5429-R / GACTTGAGCCTATCCGCCTC
AGAGTCGCCTCTGTGATCCT
comp15524_c0_seq1 / 15524-F
15524-R / CCGGGATCAAACCATGTGGA
GGTGGCTGGCAGGTAAATCT
Comp5716_c0_seq2 / 5716-F
5716-R / TTGCCATACGATGGCTTGGT
ATTGATGGGCTCAGGGTTGG
comp13823_c0_seq1 / 13823-F
13823-R / CTCAAAATGCGGCTTCACCC
CCTGGATCTCGTCGTTGGAG
comp14382_c0_seq1 / 14382-F
14382-R / CGAGTCTACCCAACGCAAGT
AGGGAGCAACGAAACGAGAG
comp2367_c0_seq1 / 2367-F
2367-R / ATAACCGTTGACCGGCCATT
AAGATGAGTTTCCGCGGTGT
comp12623_c0_seq4 / 12623-F
12623-R / GTGTTTTGGGTGGCGAGAAC
CTATCGACACCGCCTACACC
comp13666_c0_seq1 / 13666-F
13666-R / CGGCGACTATGTCGGATTGA
TGTCAGCGAACTACACGCTT
comp6499_c0_seq4 / 6499-F
6499-R / TGCTCCGATGAGGGAGGTTA
CTCGTCCACAGAAGAAGCGT
comp13878_c0_seq1 / 13878-F
13878-R / CACATCGGTGTTACTCGGCT
GGGTCGTTGTTGAGGACCAT

Location and identity of any modifications (E)

Not applied

qPCR PROTOCOL

Complete reaction conditions (E)

Reaction mixture contained 2 μL five-fold diluted cDNA template, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 10 μL FS Universal SYBR Green Master (Roche, LA, CA, USA) and 6 μL ddH2O.

Amplifications were performed in CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA), and the cycling parameters were 95°C for 3 min followed by 40 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 15 s.

Reaction volume and amount of cDNA/DNA (E)

The total reaction volume was 20 μl, volume of cDNA was 2 μl.

Primer, (probe), Mg2+ and dNTP concentrations (E)

500 nM of each primer, Mg2+ and dNTPs were parts of commercial FS Universal SYBR Green Master (Roche, LA, CA, USA), concentration unknown

Polymerase identity and concentration (E)

DNA polymerase was part of commercial FS Universal SYBR Green Master (Roche, LA, CA, USA), concentration unknown

Buffer/kit identity and manufacturer (E)

FS Universal SYBR Green Master (Roche, LA, CA, USA)

Additives (SYBR Green I, DMSO, etc) (E)

Not applied

Complete thermocycling parameters (E)

95°C for 3 min followed by 40 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 15 s

Reaction setup (manual/robotic) (D)

Manual

Manufacturer of qPCR instrument (E)

CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA)

qPCR VALIDATION

Specificity (gel, sequence, melt, or digest) (E)

Gel and melting curve analyses

For Eva Green I, Cq of the NTC (E)

No signal in NTCs or Cq > 37

Calibration curves with slope and y-intercept (E)

Not determined

PCR efficiency calculated from slope (E)

Not determined

r2 of calibration curve (E)

Not determined

Linear dynamic range (E)

Not determined

Cq variation at limit of detection (E)

Not determined

Evidence for limit of detection (E)

Not determined

If multiplex, efficiency and LOD of each assay (E)

not applied

DATA ANALYSIS

qPCR analysis program (source, version) (E)

CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA)

Method of Cq determination (E)

2nd max derivate

Outlier identification and disposition (E)

Not determined

Results of NTCs (E)

No signal in NTCs or Cq > 37

Justification of number and choice of reference genes (E)

Description of normalization method (E)

Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3

Number and stage (RT or qPCR) of technical replicates (E)

Distribution of technical replicates: 1 RNA extraction; 1 RT-PCR; 1 qPCRs

Repeatability (intra-assay variation) (E)

Not determined

Statistical methods for results significance (E)

t-test considering the P-value lower than 005

Software (source, version) (E)

Microsoft Office Excel 2007

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