Have you tried Trizol Method?

@Aymen Arif. No, i did not.

We were trying to use a method "less harmful" to the environment. But it's seems is not gonna work..

Any protocol suggestions?

3.1. RNA Extraction 1. Tissues should ideally be collected fresh and stored in liquid nitrogen. Routinely samples are collected on ice and transported for freezing within 30 to 60 min. 2. Tissues are disaggregated using a Braun-micro dismembranator. Teflon vessels and steel ball bearings are cooled in liquid nitrogen before use. Frozen tissue (50–500 mg) is placed in the vessel with a single ball bearing and agitated at 1000 cycles/second for 60 s. The vessel is then re-cooled in liquid nitrogen. This process is repeated until tissue is powdered (usually 2×; see Note 1). 3. Immediately after disaggregation of tissue, tissue material is resuspended while frozen by adding 1.5 mL of LiCl/Urea and transfer to a separate tube. The vessel is washed a further 2× with 1.5 mL of LiCl/Urea and the washing combined with the original sample. The resuspended medium is made up to 6 mL in LiCl/Urea and sonicated for 2× 30 s at maximum power using a probe sonicator. The sonicated samples are stored overnight at 4°C (see Note 2). 4. Centrifuge at 15,000g, 4°C for 30 min. The supernatant is discarded and the pellet washed with a further 6 mL of lithium chloride/urea, recentrifuged (15,000g, 4°C for 30 min) and the supernatant again discarded. 5. The pellet is resuspended in 6 mL of Tris-HCl/SDS with 50 µg/mL proteinase K (Boehringer Mannheim, UK) and incubated at 37°C for 20 min. 6. Samples are extracted with 100% phenol, followed by phenol:chloroform:isoamyl-alcohol. After each extraction, the sample is centrifuged at 2000g at room temperature for 10 min and the aqueous phase recovered. 7. After the final extraction, 300 µL of 8 M LiCl and 2.5 volumes absolute alcohol are added and samples stored at –20°C for 30 min overnight. RNA is pelleted by centrifugation at 4000g, 4°C for 45 min. The supernatant is discarded and the RNA pelleted dried and resuspended in DEPC-treated distilled water. Concentrations are estimated by optical density at 260/280 nm

Aymen Arif thanks for your answer!