M mlv h reverse transcriptase

Cells were harvested from cultures cultivated for 4, 7, 12, 16, 18, and 24 h by centrifugation at 3,300 g at 4°C for 6 min, pellets were homogenized with 1 ml of lysis solution Extract-All (Eurobio) according to manufacturer's guidelines. After that, 200 μl of chloroform were added to the suspension allowing separation of cell components. Water phase containing RNA was removed, precipitated with 500 μl of isopropanol and dissolved again in 50 μl of RNase-free water after washing in 75% cool ethanol. Samples were then treated with DNase I (Sigma-Aldrich) and purified using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Possible DNA contamination was detected by PCR detecting house-keeping gene flaA (Nachamkin et al., 1993 (link)). The integrity of RNA was verified using the Experion System (Bio-Rad). Its concentration and purity was measured using NanoDrop 2,000 (Thermo Scientific).
For reverse transcription 100 ng of RNA were mixed with 0.5 μl of 1 μM Random Hexamer Primer (Promega) and 4.5 μl of RNase-free water. This mixture was incubated 5 min at 70°C followed by 5 min on ice. After that 15 μl of Master Mix were added to mixture (5 μl of 5 × RT buffer, 1 μl 25 mM dNTP and 1 μl of M-MLV H⁻ reverse transcriptase; Promega) and it was incubated 10 min at room temperature, 50 min at 48°C and finally 15 min at 70°C to stop the reaction.

MRP1-mRNA silencing was studied using a BioRad CFX Connect QRT-PCR detection system with a SYTO9 reagent to detect the level of MRP1-mRNA expression. In summary, tissue sections were homogenised using TRI Reagent^® (Sigma-Aldrich) (1 ml per 50–100 mg of tissue). TissueLyser II Qiagen (QIAGEN^®, Hilden, Germany) was used to disrupt and homogenise tissue. Total mRNA was extracted using TRI Reagent, according to the manufacturer’s protocol. cDNA was synthesised from mRNA using M-MLV H (−) reverse transcriptase (Promega Corporation, Alexandria, NSW, Australia). The coincident measurement of the “housekeeping” gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to control the experimental variations of RNA and to normalise the MRP1-mRNA expression data, which was calculated using the ΔΔCt method. The primers were designed via the web tool (http://www.ncbi.nlm.nih.gov). All primers used for QRT-PCR of MRP1-mRNA are shown in Table 1.Table 1

Sequences of the primers used in qRT-PCR analysis

Primer ID
MRP1_Human	Forward	5′ → AAGGAATGCGCCAAGACTAG → 3′
	Reverse	5′ → CCTTAAACAGAGAGGGGTTC → 3′
GAPDH_Human	Forward	5′ → GTGAAGGTCGGAGTCAACGG → 3′
	Reverse	5′ → TGGAGGGATCTCGCTCCTGG → 3′
MRP1_Mice	Forward	5′ → TGCAGAGGCATCTCAGCAACTC → 3′
	Reverse	5′ → TTCGGCTATGCTGCTGTGTT → 3′
GAPDH_Mice	Forward	5′ → CGACTTCAACAGCAACTCCCACTCTTCC → 3′
	Reverse	5′ → TGGGTGGTCCAGGGTTTCTTACTCCTT → 3′

RNA was extracted from individual larvae using the RNeasy Mini Kit (Qiagen). cDNA was obtained using M-MLV H- reverse-transcriptase (Promega) with a dT17 primer. Quantitative PCR was performed on an ABI7300 thermocycler (Applied Biosystems) using Takyon ROX SYBR 2X MasterMix (Eurogentec) in a final volume of 10 μl. Primers used: ef1a (housekeeping gene used for normalization): GCTGATCGTTGGAGTCAACA and ACAGACTTGACCTCAGTGGT; il1b: GAGACAGACGGTGCTGTTTA and GTAAGACGGCACTGAATCCA; tnfa: TTCACGCTCCATAAGACCCA and CAGAGTTGTATCCACCTGTTA; ifng-1-1: ACCAGCTGAATTCTAAGCCAA and TTTTCGCCTTGACTGAGTGAA; ifng-2: GAATCTTGAGGAAAGTGAGCA and TCGTTTTCCTTGATCGCCCA.

Total RNA was isolated from 30 mg kidney tissue or from 5 × 10⁶ mononuclear leukocytes harvested from the blood vessels of control kidneys, isografts, or allografts on day 9 or 42 after transplantation. RNA extraction was performed using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the instructions of the supplier. One μg of total RNA was reversely transcribed using the M-MLV H⁻ reverse transcriptase and 1 μg of random hexamer primers (Promega, Mannheim, Germany). The reaction was carried out at 40°C for 1 h.

RNA was extracted from individual larvae using RNeasy^® Mini Kit (Qiagen). cDNA was obtained using M-MLV H- reverse-transcriptase (Promega) with a dT₁₇ primer or a random nonamer (for host and bacterial transcripts, respectively). Quantitative PCR was then performed on an ABI7300 thermocycler (Applied Biosystems) using Takyon™ ROX SYBR^® 2× MasterMix (Eurogentec) in a final volume of 25 µl. The following pairs of primers were used:

ef1a (housekeeping gene used for normalization): GCTGATCGTTGGAGTCAACA and ACAGACTTGACCTCAGTGGT

ifnphi1 (secreted isoform): TGAGAACTCAAATGTGGACCT and GTCCTCCACCTTTGACTTGT

il1b: GAGACAGACGGTGCTGTTTA and GTAAGACGGCACTGAATCCA

il10: CATAACATAAACAGTCCCTATG and GTACCTCTTGCATTTCACCA

tnfa: TTCACGCTCCATAAGACCCA and CAGAGTTGTATCCACCTGTTA

mmp9: AACCACCGCAGACTATGACAAGGA and GTGCTTCATTGCTGTTCCCGTCAA

E1-SINV: GACAACATGCAATGCAGAATG and CTAGTCAGCATCATGCTGCA

il22: TGCAGAATCACTGTAAACACGA and CTCCCCGATTGCTTTGTTAC

cxcl8a: GTCGCTGCATTGAAACAGAAAGCC and CTTAACCCATGGAGCAGAGGGG

il23a: CTGAAAGTGCTTAAGGAATCGG and GAGAAGGAGTAGAGTCTTTCCAC

ifng1r: ACCAGCTGAATTCTAAGCCAA and TTTTCGCCTTGACTGAGTGAA

dram1: CCTGGTTATCTGGTCATCGA and CATGAATCCAAACACACAGCT

DsRed: CAAGGAGTTCATGCGCTTC and TACATCCGCTCGGTGGA

Total RNA was isolated from tuber and leaf material using the Plant RNeasy Mini kit (Qiagen, South Africa), as described by the manufacturer. The complementary DNA (cDNA) template was obtained via reverse transcription of 1 μg of total RNA, using an oligo (dT₁₅) primer and M-MLV (H-) reverse transcriptase (Promega, Anatech, South Africa) according to the manufacturer’s instructions. The semi quantitative-PCRs (sq-PCR) were conducted with GoTaq^® DNA polymerase (Promega) in a 50-μl reaction (3 μl cDNA, 1.25 U DNA polymerase, 5× green PCR buffer, 0.5 mM of each dNTP, and 0.5 μmol of each primer) for 24 cycles, with a primer annealing temperature optimum of 58°C for all respective sq-PCR primer pairs. The constitutively expressed gene, β-tubulin2 (Nicot et al., 2005 (link); NCBI accession number 609267), was used to determine the number of cycles used for the sqRT-PCR, where expression occurred in the linear range. The Stβ-tubulin2, StLSF1, StLSF2, and StSEX4 primer pairs (StTUBFWD 5′ATGAGAGAAATCCTTCACATTC, StTUBREV 5′ GTCAGACACCTTTGGAGAAG; StLSF1FWD 5′ ATGTGCGTAATGGCCTC, StLSF1REV 5′ CCCCAGTTGAAGAGTCACTG; StLSF2FWD 5′ ATGAGAGCTCTCTGGAACTCC, StLSF2REV 5′ CCTTTTCCTTCTGAAATCGC; StSEX4FWD 5′ ATGAATTGCCTTCAGAATCTTC, StSEX4REV 5′ TGATGGCATTGTTCAGTAGT) were designed to amplify fragments of 0.5 kb from the cDNA.