Are your sperm samples difficult to lyse? Do you extract starting from whole specimen or do you proceed to prior centrifugation?
I'd recommend that you resuspend the cells you want to process in ~600 ul of β-ME -supplemented lysis buffer and incubate for 30 min @68ºC. Occasional vortexing during the incubation is welcome. Following this step, I'd recommend homogenization with a QIAshredder column. Good luck.
Not at all. I do not perform RNA isolation myself, but some colleagues do. They simply use Tryzol, obtaining a good yield. The procedure varies, I think that you should remove seminal plasma (is this what you term "whole specimen"?). However, it depends on what do you want to do... you may check some papers carrying out similar experiments.
Pure sperm RNA can be tricky. If you are using human semen then you really should remove the contaminating cell types that are present. With very few contaminating cells you can completely overwhelm the signal from the sperm, due to the low levels of RNA found in sperm. Look for the procedure from Steve Krawets lab for somatic cell lysis and sperm purification. After that, there are many methods for collection of the RNA, but use a shredding/homogenizing step to disrupt the sperm membranes.
Benjamin is right, and I forgot to mention it. We use a somatic cells lysis buffer prior to do anything with spermatozoa. I briefly collected spermatozoa from ruminant epididymes for RNA extraction, several years ago, and the procedure was really dirty (erythrocytes, epithelial cells, etc.). After suspending the sample in the lysis buffer and washing a couple of times, we could only find spermatozoa in the sample.
I thank you all for your replies. Actually, I asked these questions to Aletheia Souza (that I hope will follow this debate) but I agree with you about the protocol. They all seems reasonable procedures to follow. For 'difficulty to lyse' I meant just the disruption of sperm membrane and for 'whole specimen' I meant the seminal fluid, yes. It is reasonable that a centrifugation/washing would lead to a better starting material for extraction and less 'foreign' cellular contaminations.
If you are using human samples, sperm cell purification via gradient centrifugation (try Puresperm) removes most somatic contaminants (particularly if you are using normozoospermic samples). If the samples are more contaminated with other cell types, I would recommend an additional purification step via FACS. With FACS you can perform negative selection of leukocytes using the CD45 marker and further specify the sperm population by voltage gating. I find this approach more robust than the "somatic cell lysis buffer" protocol, since in conceptual terms I have difficulty in understanding what is fundamentally different between the cell membrane of sperm and of any other cell as to allow a targeted lysis under standard salt conditions.
I'm doing DNA extractions from sperm and it's necessary to add DTT to the lysis buffer to lyse the sperm cells because they have strong sulphide bonds in the membrane which do not break open in normal lysis buffers
I am following the trizol manual protocol but I am not attaining so good concentration .But I found the purity and concentration of frozen or thawed semen is better to extraction RNA than the fresh semen samples.
I finally stabilized total RNA extraction protocol from semen with some modification in manual trizol protocol.. I have got very good results regarding purity and concentration here I am attaching the protocol that I have used ..All the best..
Most RNA in sperm in my experience is in the midpiece and this region is not difficult to lyse (unlike the head) so as Krishna suggested, trizol followed by vigorous vortexing does most of the business. Sometimes we do sonicate to improve the efficiency (but at the cost of high DNA contamination). I have had poor yields with entire qiagen system. The larger problem in sperm RNA business is contamination from somatic cells.
Hi all,
Is there any updates of extracting RNA from human sperm? I am using total RNA purification kit from Norgen which is very good in saving time, no phenol or chloroform: 30minutes only for the whole procedure, and high purity. The problem is the quantity ranging from sample to sample which could affect downstream work and the best quantity was 130ng/ul.
Hi everybody,
I'm trying to obtain RNA-seq data from some ant sperm samples. Samples have been in -80. The good thing about these ejaculates is that they don't seem to have other cell types in them, I've checked this via FACS, so I wouldn't need to use a somatic cell purification step. However, my experimental samples are only about 8-10 microliters of ejeculate each (and I would not be able to pool them to increase the starting material as they are part of a manipulative experiment), and I'm now trying to obtain enough yield and quality for RNA-seq to work fine, but until now I haven't had that great success with the extraction methods that I've tried. I've used the RNeasy kit and the RNeasy Mini universal kit (which is supposed to be more similar to Trizol, as it has Qiazol), but both gave very low yields and quality (about 100-300 pg/ul and RQI of about 3 as measured with the Experion RNA HighSens kit). I don't have many additional samples to try different extraction protocols, so I hope someone could give me some suggestions on what to try, or if you think I should loose hope of making this project work since the starting amounts of sperm are not that much.
Thank you very much for your help!
Joanito
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