RNeasy PowerMicrobiome Kit (50)

Protocol tips

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	Lysis by TRIzol LS reagent, increases the input volume for RNA extraction, which contributes to minimize the SLOD, reducing the sample processing time compared to TRIzol.

Protocol tips
Lysis by TRIzol LS reagent, increases the input volume for RNA extraction, which contributes to minimize the SLOD, reducing the sample processing time compared to TRIzol.

Publication protocol

Refinement of PEG+TRIzol by RNeasy PowerMicrobiome Kit: The PEG + TRIzol method was slightly modified by combining TRIzol LS reagent (Thermo Fisher Scientific) with RNeasy PowerMicrobiome Kit (QIAGEN) (TRIzol + RNeasy PowerMicrobiome Kit). This method contains two steps: sample lysis by TRIzol LS reagent and RNA precipitation, purification and elution using RNeasy PowerMicrobiome Kit. The theoretical advantages of this method are to increase the input volume for RNA extraction, which contribute to minimize the SLOD, and to reduce the sample processing time compared to TRIzol.
Specifically, a 250 μL aliquot of PEG concentrate was mixed with 750 μL of TRIzol LS reagent and vigorously vortexed for 30 s. After a 5 min incubation, 200 μL of chloroform was added to the mixture and vigorously vortexed for 15 s. The mixture was then centrifuged at 12,000g for 15 min at 4 °C. A colorless upper aqueous phase was mixed with 150 μL of solution IRS, which is designed to effectively remove PCR inhibitors (Ahmed et al., 2020b). Thereafter, the RNA was precipitated and filtered through a silica-based column followed by washing and elution, as described in the manufacturer's instructions, without DNase treatment. The final volume of eluted RNA was 60 μL. Thus, the concentration factor of this process was 4.2-fold.
The applicability of PEG + TRIzol + RNeasy PowerMicrobiome Kit was confirmed by raw sewages A, B, and C. The whole process recovery of φ6 was evaluated in duplicate for each raw sewage. The RT-qPCR process follows method 1 (two-step RT-qPCR using High-Capacity cDNA Reverse Transcription Kit and TaqMan™ Gene Expression Master Mix).

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Manufacturer protocol

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