5×105 BMDMs or neutrophils were plated on 24-well tissue culture treated plates. Cells were lysed with TRIzol (Invitrogen) and RNA extraction was carried out according to the manufacturer’s instructions. Reverse transcription was performed using M-MuLV reverse transcriptase, RNase inhibitor, random primers 9, and dNTP mix (New England BioLabs) to synthesize cDNA. cDNa was analyzed for relative mRNA levels using SYBR Green (Applied Biosystems) and intron spanning primers. GAPDH was used to normalize mRNA levels. Post-amplification melting curve analysis was performed to confirm primer specificity.
Random primers 9
Manufactured by New England Biolabs
Sourced in United States
Random primers are short, synthetic DNA sequences that are designed to bind to multiple regions within a target DNA molecule. They are commonly used in various molecular biology techniques, such as polymerase chain reaction (PCR) and reverse transcription-PCR (RT-PCR), to amplify unknown or unpredictable DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
M mulv reverse transcriptase, by New England Biolabs (3 mentions)
Sybr green, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by New England Biolabs (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Dntp mix, by New England Biolabs (1 mentions)
Thermopol, by New England Biolabs (1 mentions)
Dnase 1, by Merck Group (1 mentions)
3 protocols using random primers 9
1
Quantifying mRNA Levels in Myeloid Cells
2
RNA Extraction and RT-qPCR Analysis
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The 5 × 105 BMDMs were plated on 24-well tissue-culture-treated plates. Cells were lysed with TRIzol (Invitrogen), and RNA extraction was carried out according to the manufacturer’s instructions. Reverse transcription was performed using M-MuLV reverse transcriptase, RNase inhibitor, random primers 9, and deoxyribonucleotide triphosphate mix (New England BioLabs) to synthesize complementary DNA (cDNA). cDNA was analyzed for relative mRNA levels using SYBR Green (Applied Biosystems) and intron spanning primers. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize mRNA levels. Postamplification melting curve analysis was performed to confirm primer specificity.
3
Transcriptional Analysis of Mycobacterial Efflux Pumps
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Total RNA from M. smegmatis was isolated as previously described [24 ], from bacteria grown to OD600nm 0.5. Residual DNA in the mycobacterial total RNA preparations was removed using DNase I according to the manufacturer's instructions (Sigma-Aldrich, Inc., Darmstadt, Germany). A control with no reverse transcriptase (NRT) was used for a PCR reaction with primers to 16S rRNA [25 (link)] (Table 2 ) to confirm complete DNA removal. The M-MuLV Reverse Transcriptase (New England Biolabs, MA, USA) and random primers 9 (New England Biolabs, MA, USA) (Table 2 ) were used to produce cDNA according to the manufacturer's instructions. The cDNA was used to amplify the mfp genes, in triplicate, using ThermoPol® (New England Biolabs, MA, USA) and specific primers (see Figure 1 and Table 1 : mfpED, mfpDC, mfpCB, mfpBA, and mfpA). After PCR amplification, the fragments were sequenced with the amplification primers in the forward and reverse directions (Macrogen, Korea).
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