[PDF] SUPPLEMENT S2: RT-QPCR METHODS - Free Download PDF (2024)

1 SUPPLEMENT S2: RT-QPCR METHODS Table S2.1 Experimental design Definition of experimental and control groups Number in ...

SUPPLEMENT S2: RT-QPCR METHODS Table S2.1 – Experimental design Definition of experimental and control groups Number in each group Assay carried out by core or investigator's laboratory

Experimental: Madison (MSN) mouse strain. Control: outbred hsd:ICR (ICR) mouse strain. 8 MSN, 8 ICR. All mice were male.

Authors’ contributions to qPCR section

C. Michael Saul: all molecular work, half of dissection work, writing. Griffin M. Gessay: half of dissection work. Stephen C. Gammie: bred mice, provided funding and lab space, writing.

Carried out in investigator's laboratory.

Table S2.2 – Tissue Samples Description Volume or Mass of sample Dissection Type Processing Procedure If frozen, how quickly? Sample storage conditions

Fresh frozen whole hippocampus. See table S2.10. Gross dissection of hippocampal tissue from brain. Animals were euthanized by cervical dislocation under isoflurane anesthetic, decapitated, and their hippocampi were immediately dissected from their brains. Samples frozen immediately on dry ice upon dissection. Stored at -80°C for no more than 12 weeks prior to RNA extraction.

Table S2.3 – Nucleic Acid Extraction Procedure and/or instrumentation Name of kit and details of any modifications Sources of additional reagents used

Mortar and pestle disruption, guanidinium thiocyanate-phenol-chloroform extraction, and spin column cleanup and purification. Bio-Rad Aurum Total RNA Fatty and Fibrous Tissue Kit (catalog number 7326830) used according to the manufacturer’s specifications. Chloroform (Acros Organics, catalog number AC42355-0250) Ethanol (Fisher Scientific, catalog number BP2818-500)

Details of DNase treatment

On-column treatment with DNase I according to manufacturer's specifications.

Contaminaion assessment of input RNA

NanoDrop curves used to assess presence of presence of protein, salt, and organic contaminants. All curves indicated clean samples.

Nucleic acid quantification

See table S2.10.

Instrument and method of nucleic acid quantification

NanoDrop spectrophotometer, absorbance at 260nm.

Purity (A260/A280)

See table S2.10.

Yield

See table S2.10.

RNA integrity instrument

Agilent RNA 6000 Nano Chips with Agilent BioAnalyzer 2100.

RIN

See table S2.10.

Inhibition testing

Cq dilution, 1:8 using Ywhaz. As expected, diluted samples ran ~3 cycles behind undiluted samples.

Table S2.4 – Reverse Transcription Complete reaction conditions Amount of RNA and reaction volume Priming oligonuclotide and concentration Temperature and time Manufacturer of reagents and catalog number Cq with and without RT Storage conditions of cDNA

500µM dNTP mix, 20mM Tris-HCl (pH 8.4), 50mM KCl, 5mM MgCl2, 2.5mM dT 20mers, 10mM DTT, 2U/µL RNaseOUT, 10U/µL SuperScript III RT. 25µL reactions, 2µg total RNA used in each reaction. 1.25µL RNAse H added after reaction termination. oligo-dT 20mers, final reaction concentration of 2.5mM. Prior to cDNA synthesis, RNA, primers, and dNTPs were denatured together at 65°C for 5 min. cDNA synthesis took place at 50°C for 50 min followed by an 85°C reaction termination step for 5 min. After reaction termination, the RNase reaction ran at 37°C for 20 min. Invitrogen SuperScript III First-Strand Synthesis System for RT-PCR (catalog number 18080-051) See table S2.10 for no RT Cqs. While some gDNA contamination is present, its effect on experiments are negligible and stochastic according to hypothesis testing on no RT controls. This contamination is mostly a source of random error. Stored at -80°C for no longer than 6 months.

Table S2.5 – qPCR Target Information Gene symbol

See table S2.11.

Accession number

See table S2.11.

Location of amplicon

See table S2.11.

Amplicon length

See table S2.11.

In silico specificity

All primers screened for specificity using NCBI Primer-BLAST.

hom*ologs amplified

No primers amplified pseudogenes or retropseudogenes.

Sequence alignment

Aligned in NCBI Primer-BLAST.

Location of each primer by exon or intron

See table S2.11.

Targeted splice variants

Each primer set targets all splice variants for every transcript of interest as they are documented in the NCBI RefSeq RNA database.

Table S2.6 – qPCR oligonucleotides Primer sequences

See table S2.11.

Probe sequences

Not applicable; dsDNA binding dye chemistry used.

Location and identity of any modifications Manufacturer of oligonucleotides Purification method

No modifications. UW-Madison Biotechnology Center DNA Synthesis Facility. Standard desalting and lyophilization.

Table S2.7 – qPCR protocol Complete reaction conditions

2X Bio-Rad SsoFast EvaGreen Super Mix without ROX (catalog number 1725204) used with no modifications.

Reaction volume and amount of cDNA/DNA

20µL reactions; 2µL 1:5 diluted cDNA used in each reaction.

Primer Concentration

500nM forward and 500nM reverse primer for all primer sets.

Mg2+concentration

3.0mM MgCl2.

dNTP concentration

200µM each of dATP, dTTP, dCTP, and dGTP. 800µM dNTP total.

Polymerase identity

Bio-Rad SsoFast Taq Fusion Polymerase.

Polymerase concentration

Proprietary concentration.

Buffer identity and manufacturer

Bio-Rad qPCR buffer provided with SsoFast EvaGreen Supermix.

Exact buffer chemistry

Proprietary composition.