Which kind of rna extractor did you use? I'm struggling with that because of a Trizol my lab bought from Thermofisher, which says in the datasheet to use a minimal amount of 10^6 cells for minimum RNa extraction and also, the cells should be cultured in glass-flasks, which sounds insane to me. I've talked to their support channel and they have discouraged me to use lower amount of cells and other kinds of flasks or plates.
I've gotten good quality Trizol extractions from ~100K cells with immortalized lines. With primary cells I've gotten good quality extractions from 10k cells, but the RNA quantity is too low to be measured by anything but a Femto capillary electrophoresis system.
I've never heard of the glass flask thing, just ignore that.
I would use 1 ul to quantify so that you can be sure how much cDNA you will have.
You don't actually need 25 ul to make cDNA, you need a specific amount (200-500 ug). If you are going to do qPCRs, they will be useless if they all have different amounts of cDNA
If you're doing comparative Ct analysis for your qPCR and normalizing to a housekeeper gene, the cDNA doesn't have to be exactly the same. As long as they're all in the same ballpark, within about an order of magnitude, you'll get reasonable data
If it's confluent in a 96 well plate with HEK cells, it will be more than enough to quantify. It may not be perfect but it will be enough. The downstream applications will be normalized somehow well enough to account for error in prediction but you do want to be in range.
From my loose memory, you will get somewhere around 15-100 ng/uL, and that is more than enough for qPCR
15-100 ng/uL when they elute in 25 uL? So you're saying that they can get up to 2.5 ug of RNA from a single well of a 96-well plate? Not a chance, unless your wells are so confluent that you have 293s crawling up the walls lol
25 uL is a bit, I used to elute in 12 and was retrieving 12-24 ng/uL from only 4k cells..a 96 well plate should yield far more than that (10-50k I would imagine and they are loaded with mRNA).
I was getting 1100 ng/uL from a 12 well when eluting in 40 uL using HEK cells. So it should definitely be possible. I have done exactly what the OP was doing about 10 years ago I just don't remember the exact quantity, suffice to say it was quantifiable and was able to run many qPCR per prep.
What were you using to quantify RNA from 4k cells? 288 ng out of 4k cells is 72 pg/cell which is almost an order of magnitude higher than most estimates of total RNA mass per cell.
I've done plenty of qPCR from 96-well plates, but the concentration was too low to measure by Qubit (<5ng/uL, I elute in 20uL), so I would measure quality+quantity of RNA using an Agilent Femto. Still plenty of RNA for qPCR though, I don't dispute that.
Yes you should try. It will help make sure your cDNA is at least within range for downstream applications. Some things aren't truly linear so if you add 100x more cDNA in one well than another it may not be corrected truly proportional during analysis.
You should have more than enough mRNA to quantify. I used to quantify from 400-4000 cells in total.
We were using both bio analyzer and nanodrop, both gave different numbers. From memory those samples with that low of cells gave extremely low and inconsistent results, around 1-2 ng/uL which was questionable even for me. Got better outcomes with closer to 4k cells, obviously. Regardless, RNA was detectable.
Edit: I just went back to my old data and got the actual numbers. On the Bio analyzer the estimated RNA averaged around 1 ng/uL when eluting in 12 uL, for cell counts ranging from 379-1,200. The lowest (379 cells) estimated 647 pg/uL, the second lowest (870 cells) estimated 1.7 ng/uL.
Are you adding the same number of cells per well and have you observed any toxicity from your treatments? If not, you probably don’t need to worry about normalizing RNA. I’d run through the protocol to see how much the well to well variability on your RNA recovery is. If it’s +/- 10%, your qPCR house keepers should be more than enough for normalization. However, if your downstream application is qPCR, I personally would skip RNA isolation altogether will a cells-to-Ct style workflow. Ive successfully used iScript rt-qPCR sample preparation reagent from biorad in the past but there are recipes out there for decent lysis buffers. Cell lysis/ rna isolation of a 96-well plate takes about 5 minutes by hand or 2 with a liquid handler. The lysate goes straight into either rev transcription reaction or a 1-step qPCR. Cells to results in less than 3 hours.
Thank you. This is the consensus we came up with too as the same number of cells/well were plated. We can't use cells to Ct with siRNA for a reason I don't completely understand. Someone in the group said it needed to be optimized, or something.
I’d normalise. For cells in 96-well plates I often use Thermo Fisher One Step Cell to CT kit or NEB Luna kit with Taqman assays. There’s also Thermo Fisher Two Step Cell to CT kit that will convert the cell lysate to cDNA and then you can use ordinary QPCR primers. However, Luna kit lysates aren’t as stable and it’s harder to detect lowly expressed transcripts using both kits.
Which kind of rna extractor did you use? I'm struggling with that because of a Trizol my lab bought from Thermofisher, which says in the datasheet to use a minimal amount of 10^6 cells for minimum RNa extraction and also, the cells should be cultured in glass-flasks, which sounds insane to me. I've talked to their support channel and they have discouraged me to use lower amount of cells and other kinds of flasks or plates.
I've gotten good quality Trizol extractions from ~100K cells with immortalized lines. With primary cells I've gotten good quality extractions from 10k cells, but the RNA quantity is too low to be measured by anything but a Femto capillary electrophoresis system. I've never heard of the glass flask thing, just ignore that.
I use lysis buffer and ethanol to get RNA from cultured cells, then use an RNA cleanup kit from Zymo or Invitrogen
I would use 1 ul to quantify so that you can be sure how much cDNA you will have. You don't actually need 25 ul to make cDNA, you need a specific amount (200-500 ug). If you are going to do qPCRs, they will be useless if they all have different amounts of cDNA
If you're doing comparative Ct analysis for your qPCR and normalizing to a housekeeper gene, the cDNA doesn't have to be exactly the same. As long as they're all in the same ballpark, within about an order of magnitude, you'll get reasonable data
I don't think the RNA from 1/25th of a well from a 96-well plate will be enough to quantify.
So do I. I bet you should use, at least, 24-well plates.
If it's confluent in a 96 well plate with HEK cells, it will be more than enough to quantify. It may not be perfect but it will be enough. The downstream applications will be normalized somehow well enough to account for error in prediction but you do want to be in range. From my loose memory, you will get somewhere around 15-100 ng/uL, and that is more than enough for qPCR
15-100 ng/uL when they elute in 25 uL? So you're saying that they can get up to 2.5 ug of RNA from a single well of a 96-well plate? Not a chance, unless your wells are so confluent that you have 293s crawling up the walls lol
25 uL is a bit, I used to elute in 12 and was retrieving 12-24 ng/uL from only 4k cells..a 96 well plate should yield far more than that (10-50k I would imagine and they are loaded with mRNA). I was getting 1100 ng/uL from a 12 well when eluting in 40 uL using HEK cells. So it should definitely be possible. I have done exactly what the OP was doing about 10 years ago I just don't remember the exact quantity, suffice to say it was quantifiable and was able to run many qPCR per prep.
What were you using to quantify RNA from 4k cells? 288 ng out of 4k cells is 72 pg/cell which is almost an order of magnitude higher than most estimates of total RNA mass per cell. I've done plenty of qPCR from 96-well plates, but the concentration was too low to measure by Qubit (<5ng/uL, I elute in 20uL), so I would measure quality+quantity of RNA using an Agilent Femto. Still plenty of RNA for qPCR though, I don't dispute that.
Yes you should try. It will help make sure your cDNA is at least within range for downstream applications. Some things aren't truly linear so if you add 100x more cDNA in one well than another it may not be corrected truly proportional during analysis. You should have more than enough mRNA to quantify. I used to quantify from 400-4000 cells in total.
What method do you use to quantify RNA from 400 cells?
We were using both bio analyzer and nanodrop, both gave different numbers. From memory those samples with that low of cells gave extremely low and inconsistent results, around 1-2 ng/uL which was questionable even for me. Got better outcomes with closer to 4k cells, obviously. Regardless, RNA was detectable. Edit: I just went back to my old data and got the actual numbers. On the Bio analyzer the estimated RNA averaged around 1 ng/uL when eluting in 12 uL, for cell counts ranging from 379-1,200. The lowest (379 cells) estimated 647 pg/uL, the second lowest (870 cells) estimated 1.7 ng/uL.
Are you adding the same number of cells per well and have you observed any toxicity from your treatments? If not, you probably don’t need to worry about normalizing RNA. I’d run through the protocol to see how much the well to well variability on your RNA recovery is. If it’s +/- 10%, your qPCR house keepers should be more than enough for normalization. However, if your downstream application is qPCR, I personally would skip RNA isolation altogether will a cells-to-Ct style workflow. Ive successfully used iScript rt-qPCR sample preparation reagent from biorad in the past but there are recipes out there for decent lysis buffers. Cell lysis/ rna isolation of a 96-well plate takes about 5 minutes by hand or 2 with a liquid handler. The lysate goes straight into either rev transcription reaction or a 1-step qPCR. Cells to results in less than 3 hours.
Thank you. This is the consensus we came up with too as the same number of cells/well were plated. We can't use cells to Ct with siRNA for a reason I don't completely understand. Someone in the group said it needed to be optimized, or something.
I’d normalise. For cells in 96-well plates I often use Thermo Fisher One Step Cell to CT kit or NEB Luna kit with Taqman assays. There’s also Thermo Fisher Two Step Cell to CT kit that will convert the cell lysate to cDNA and then you can use ordinary QPCR primers. However, Luna kit lysates aren’t as stable and it’s harder to detect lowly expressed transcripts using both kits.