Hi Elahe

Just you need to monitor few steps  carefully of Trizol manual method  to get good result

First, crush that creature (mature 3-5 will be enough) with trizol either with a micropestle or by a homogenizer. The volume of trizol will be 750-1000uL.

All the centrifugation should be done @ 4 degrees.

Then centrifuge (10000 rpm for 10 min) to pellet the debris, supernatant will be separated. Wait for 5 min @ RT.

Add 200-250uL of chloroform shake vigorously for 20 sec (Red color of trizol will be turned pinkish ) wait for 5 min @ RT.

Centrifuge @ 12000 rpm for 15 min to separate transparent sup. in a new vial.

Then add ice child (-20, 500uL) Isoprop dropwise by pipette wait to react for 5 min @ rt and incubated for overnight @ -80.

Centrifuge @ 12000 rpm for 15 min, a white pellet will be observed and then clean by 70% chilled ethanol, air dried and dissolved in 50 uL RNase-free water.

During RNA extraction each and every accessory should be RNase free and try to use a dust free enviornment for better result.

Hope will help.