If you're looking for an alternative method, have you thought about using 15, 18 and 21-gauge needles to pass your sample through? I've used this method for nearly 100 left venricular samples that were stored in RNAlater which rubberized them. Got good yield RNA yields and integrity with them, but I wouldn't recommend for protein.

I have tried the needle method- did tou also use a motor homogenizer on it after or only the needle? I found that with the needle I was not able to make all my tissue dissolve in the RLT buffer so that later led to my column getting clogged. Any advice on this would be appreciated!

To get RNA, I found this to be a viable method. I was getting yields at around 100 ng/uL, however with protein this was insufficient. For protein, I used a combination of mechanical homogenization (IKA) with RIPA buffer which was sufficient with good yields. I would be surprised if this method didn't work with your tissue, the tissue I used was stored in RNAlater which made it rubbery - very difficult to break down mechanically and enzymatically. You only need a small bit of tissue, for me I sliced the (mouse heart) ventricle in 8-12 pieces and only used very few of those slices. Then you take 1-3 pieces, pass it through 15 gauge needle first a dozen or so times, and once the pressure/resistance is not there anymore, move unto 18 gauge needle. You will feel a strong pressure/resistance, carefully/slowly push the needle in and repeat this process again a dozen or so times. Then lastly you can also pass through 21 gauge which is very difficult at first but once you get used to it, not that much more of a challenge than the 18 gauge needle. The combination of 15, 18 and 21 gauge gave me great yield and quality of RNA. You can do 12-16 of that a day without too much of a hassle. I think you should try to put less tissue in, perhaps you are putting in too much tissue and they're not thoroughly homogenized when they are loaded into the columns? When you are passing through the needle, initially you should feel the challenge via resistance, if there's no more challenge after a few rounds of pass through the needle, then you need to move unto a finer needle. Always wear protective goggles with this method, at times the needle will not take the pressure and blowback fluid from the syringe end!

Can Justin Kiessling I tried this out today, although I only had 8, 21, and 25 gauge needles available to me and it seemed to work fine. However, my yields were about 10-18 ng/uL while I was getting 70-100 ng/uL on the same tissues using a motor homogenizer, which is strange. Perhaps because I've been using RLT Plus buffer and beta-mercaptoethanol those are not as compatible with the syringe and needle method. Thanks so much for your suggestion though

The reason you're getting less yield with gauge needles might be due to incomplete/partial mechanical breakdown. I see you said that you used 8 (perhaps you meant 18?), perhaps the 8 gauge isn't breaking down enough of the tissue clumps so that the 21 and 25 gauge can finish off at a finer level. I would stress that the first gauge is crucial for the success of the finer needles. 21 and 25 are quiet tough, I know 21 gauge was giving me real difficulty/strain on my fingers.