Total rna isolation kit

Total RNA was isolated using a total RNA isolation kit (Macherey-Nagel, Duren, Germany) and denatured at 70 °C for 10 min. cDNA was synthesised with oligo dT primers and a reverse transcriptase kit (Takara). RT-qPCR amplification was performed using TaqMan PCR probe sets (Supplementary Table) and a Step-One Plus real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). Target gene mRNA expression levels were normalised to β-actin mRNA expression. Data are represented as the mean ± standard deviation (n = 3).

The P. denitrificans strains were grown in M9-minimal medium containing 5 g/L sodium gluconate. The cells were cultivated under the aerobic condition at 37 °C and 200 rpm in an orbital incubator shaker. 3-HP and/or various chemicals (3-hydroxyisobutyrate, 3-hydroxybutyrate, L-valine, lactic acid, acetic acid, propionic acid, 1,3-propanediol and 2,3-butanediol) at 25 mM was supplemented at the OD₆₀₀ of ~0.4–0.5. After a further 2 h cultivation, approximately 5 × 10⁸ cells were collected and centrifuged at 5,000 × g for 10 min. The cell pellets were immediately resuspended in 500 μL of RNA solution (Ambion, UK) and RNA was extracted using a total RNA isolation kit (Macherey-Nagel, Germany). One microgram of total RNA was employed to synthesize the first-strand cDNA in a 20 µL reaction using the SuperScript III first-strand synthesis system (Invitrogen, USA). A real-time PCR analysis was performed, according to the SYBR green method, in a 20 µL reaction volume using the StepOne Real Time PCR system (Applied Biosystems, USA). The PCR efficiencies of all of the primers were experimentally determined and found to be suitable for reliable copy-number quantification. The mRNA quantity was estimated based on ΔΔCT value, as described previously²⁵ . The assays were performed in duplicate, and a template-less reaction was used as a negative control.

Total RNA was extracted from kidney tissue using the Total RNA Isolation Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Green PCR Master mix (Fast Start Universal SYBR Green Master; Roche). To quantify the mtDNA/gDNA ratio, qPCR was used to amplify one gene from the mitochondrial genome (Nd1 in humans) and one gene from the nuclear genome (β-globin in humans). Primer sequences were as follows: Nd1 forward 5′-CAA ACC GGG CCC CCT TCG AC-3′; Nd1 reverse 5′-CGA ATG GGC CGG CTG CGT AT-3′; β-globin forward, 5′-GAG AAT GGG AAG CCG AAC ATA-3′; β-globin reverse 5′-CCG TTC TTC AGC ATT TGG ATT T-3′.