DNA was extracted per manufacturers’ instructions from urine, blood, and kidney using a NucleoSpin tissue kit (Clontech). Quantification of Leptospira 16s rRNA was done using TAMRA probe and primers from Eurofins (Huntsville, AL) by real-time PCR (qPCR) (StepOne Plus). RNeasy mini kit (Qiagen) was used to extract total RNA followed by reverse transcription using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Real-time PCR on the cDNA was performed as described (8 (link)). For RT-PCR, we used TAMRA probes specific for inducible nitric oxide synthase (iNOS), Collagen A1 (ColA1), keratinocyte-derived chemokine (KC, CxCL1), macrophage inflammatory protein 2 (MIP-2, CxCL2), RANTES (CCL5), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ) and IL-10. β-actin was used as control for the comparative CT method (10 (link)).
Tamra probe and primers
Manufactured by Eurofins
TAMRA probe and primers are fluorescent molecules used in a variety of molecular biology techniques, such as real-time PCR and DNA sequencing. TAMRA (Tetramethylrhodamine) is the fluorescent dye that is attached to the probe or primer, which emits a specific wavelength of light upon excitation. These probes and primers can be used to detect and quantify target DNA or RNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Nucleospin tissue kit, by Takara Bio (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
β actin, by Eurofins (1 mentions)
Nanodrop instrument, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna blood kit, by Macherey-Nagel (1 mentions)
4 protocols using tamra probe and primers
1
Quantifying Leptospira Infection via qPCR
2
Quantification of Leptospira in Tissues
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DNA was extracted per manufacturers’ instructions from urine, blood, and kidney using a NucleoSpin tissue kit (Clontech). Quantification of Leptospira 16s rRNA was done using TAMRA probe and primers from Eurofins (Huntsville, AL) by real-time PCR (qPCR) (StepOne Plus). RNeasy mini kit (Qiagen) was used to extract total RNA followed by reverse transcription using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Real-time PCR on the cDNA was performed as described [9 (link)]. For RT-PCR, we used TAMRA probes specific for inducible nitric oxide synthase (iNOS), Collagen A1 (ColA1), keratinocyte-derived chemokine (KC, CxCL1), macrophage inflammatory protein 2 (MIP-2, CxCL2), RANTES (CCL5), tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). β-actin was used as control for the comparative CT method [14 (link)].
3
Quantification of Leptospira and Gene Expression
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DNA was extracted from blood, urine, and kidney using a NucleoSpin tissue kit. Leptospira was quantified using TAMRA probe and primers (Eurofins) to Leptospira 16s rRNA by qPCR. Total RNA was extracted from tissues using an RNeasy mini kit and reverse transcribed using a high-capacity cDNA reverse transcription kit. cDNA was subjected to real-time PCR using primer and TAMRA probes previously described [11 (link)]. PCR data are reported as the relative increase in mRNA transcript levels of CxCL1 (KC), CxCL2 (MIP-2), CCL5 (RANTES), TNF-α, IFN-γ, iNOS and ColA1 corrected for by the respective levels of β-actin or to GAPDH. A primer list is provided in supplemental material (S1 Table ).
4
Leptospira Quantification by qPCR
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Quantification of Leptospira was performed using TAMRA probe and primers (Eurofins) to leptospiral 16s rRNA (L. interrogans) and 23s rRNA (L. biflexa) by qPCR. Primers and probes are described in Table S1 Table S1
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