I am trying to extract RNA from Streptomyces sp by manual method I am unable to get any results Kindly help me to find a standardized manual protocol?

Extracting RNA from Streptomyces species can be challenging due to their complex cell walls and the presence of secondary metabolites. However, there are several manual protocols available that have been successfully used for RNA extraction from Streptomyces. Here is a standardized manual protocol that you can try:

Materials:

Tris-EDTA (TE) buffer (pH 8.0)
Lysozyme
Proteinase K
Phenol:chloroform:isoamyl alcohol (25:24:1)
Chloroform:isoamyl alcohol (24:1)
Isopropanol
Ethanol (70% and 100%)
RNase-free water
DEPC-treated water
RNase inhibitor (optional)

Protocol:

Harvest Streptomyces cells by centrifugation at 4000 rpm for 10 minutes at 4°C. Discard the supernatant and resuspend the cell pellet in TE buffer (pH 8.0).

Add lysozyme to a final concentration of 10 mg/ml and incubate the suspension at 37°C for 30 minutes to lyse the cells.

Add proteinase K to a final concentration of 100 μg/ml and incubate the suspension at 55°C for 1 hour to degrade proteins.

Add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), mix gently by inverting the tubes, and centrifuge at 12,000 rpm for 10 minutes at 4°C.

Transfer the aqueous phase (top layer) to a new tube, avoiding the interface and the organic phase.

Add an equal volume of chloroform:isoamyl alcohol (24:1), mix gently by inverting the tubes, and centrifuge at 12,000 rpm for 10 minutes at 4°C.

Again, transfer the aqueous phase to a new tube, avoiding the interface and the organic phase.

Precipitate the RNA by adding 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of cold absolute ethanol. Mix well by inverting the tubes and incubate at -20°C for at least 1 hour or overnight.

Centrifuge the tubes at 12,000 rpm for 30 minutes at 4°C to pellet the RNA.

Carefully remove the supernatant without disturbing the RNA pellet.

Wash the RNA pellet with 1 ml of 70% ethanol, centrifuge at 12,000 rpm for 10 minutes at 4°C, and carefully remove the supernatant.

Air-dry the RNA pellet for 10-15 minutes or until the ethanol evaporates completely.

Resuspend the RNA pellet in 20-50 μl of DEPC-treated water or RNase-free water.

Optional: Add RNase inhibitor to the RNA solution to protect it from degradation.

Store the RNA at -80°C or use it immediately for downstream applications such as RT-PCR, qPCR, or RNA sequencing.

This protocol should yield RNA of sufficient quality and quantity for downstream applications. However, optimization may be required based on the specific Streptomyces species and growth conditions used in your experiments. Additionally, using RNase-free reagents and equipment throughout the protocol is essential to prevent RNA degradation.

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