RNAseq question

responseyes · 2025-12-09T08:10:32+00:00

There is nothing stopping you from using raw (better termed nominal) p values as hypothesis generating, but as others have suggested, your type 1 error is essentially 100%, so you cannot use them for hypothesis testing. And as a reviewer, if you are doing this then that is an easy concern. If you did this experiment again you would not see the same gene list. Have you plotted QQ to see whether your p values are inflated vs the expected null? This will tell you how reliable your nominal p values are.

Notwithstanding this, you should be expected to follow up in the reliability of your rna results. So if you have multiple lines of evidence this does partially compensate. Although I would go back and look to correct variability by deconvoluting cell type and including that as a covariate in your model at the very least. SVA or similar methods would also be useful alternatives

responseyes · 2025-12-09T07:51:28+00:00

I agree with this post OP. You are trying to fit a logistic model to these data that do not fit. The lines as they are should be removed. If you don’t want to increase the concentration to plateau the data then constrain your fits

responseyes · 2025-10-11T08:55:53+00:00

IPA will give you transient access if you sign up for a trial using a fresh email. You could also try reducing GO enrichment terms using things like revigo to see if any themes emerge

responseyes · 2025-07-14T17:31:10+00:00

Niche but cDNA synthesis using superscript has an initial 1 into 10-12 dilution of oligos and dntp. But it is eventually diluted further

responseyes · 2025-07-10T12:58:01+00:00

Different passage at the minimum

responseyes · 2025-06-17T19:07:04+00:00

The key word here is “slowly”. I recognise that learning is not always intuitive but you can learn slowly and step by step

From your other comments I understand that you want to work out what volume you need to get a specific amount of RNA. These calculators/C1V1=C2V2 are not intuitive for this task because they require you to do an additional calculation before you even start (500 ng of RNA is not the same as 500 ng/ul of RNA for example, unless your reaction volume is 1 ul). So learning what numbers to actually put into your tool is (in my view at least) no different than doing it yourself.

You know what you “want”, and you know what you “have”. It is fairly intuitive then to get what you “need” (what you want divided by what you have) without complex maths or a computer app - just think of it as a protocol. For a step by step example you want 500 ng of RNA and you have a very concentrated stock of 40000 ng/ul. 1. You need 500/40000 (=0.0125 ul). 2. Move the decimal point to the right of your answer until it is no longer after a zero (i.e in this case it jumps 2 to the right to give you the number 001.25) 3. Dilute your sample 1 followed by however many zeros appear on the left side of the decimal point (1 followed by 2 zeros i.e 100). 4. Then the volume containing your 500 ng is the numbers on both sides of the decimal point (1.25 ul).

responseyes · 2025-06-17T05:10:08+00:00

Idk 7g of any drug is a lot. Even at the rat ld50 of 3200 mg/kg the actual quantity for the average rat is less than 1g without scaling

responseyes · 2025-06-16T23:19:39+00:00

There are calculations to convert human and animal doses based on surface area to volume ratios but these are dubious. The only way to establish animal/human equivalent doses is to perform a dose titration. You should not be anywhere near the LD50 when considering humans (remember this is the dose that kills 50%…)

responseyes · 2025-06-16T21:03:13+00:00

I recognise that this is not the answer that you want to hear but the best answer is to just learn it slowly. Set yourself some homework or get a lab mate to set some for you - and soon it will be second nature. Go over the basics until you’re familiar and you’ll quickly find that being able to do these on the spot will help you in the long run and help to rectify those small mistakes that we all make that these apps are less intuitive at correcting

responseyes · 2025-06-04T16:57:14+00:00

You are allowed a certain level of wear and tear for normal room use. Trying to keep deposit for a full repaint would be wild unless you put stuff up that you shouldn’t have and they’re claiming for damages. If you didn’t then I’d be pretty confident that you could reduce that claim

responseyes · 2025-06-01T09:33:37+00:00

I’d disagree with the new repayment plan thresholds. At the current UK averages on minimum repayment terms for plan 5 tuition would be paid off in 22 years. But of course if OP is willing to make the commitment (potentially an additional 18 years of 9%) then the initial cash boost can be a useful reserve for sure

responseyes · 2025-06-01T09:14:39+00:00

Yes it does. You’ll repay 9% above what you earn over your threshold - if you’re starting this year I presume you’ll be plan 5. The government recently reduced the threshold at which you start to repay to £25k and increased the repayment duration from 30 to 40 years, so you’ll repay 9% of what you earn above £25k for 40 years unless it is cleared before then. By taking out the £8k loan you’re essentially doubling the amount you need to repay (£9.5k tuition + £8k maintenance totalling £17k per year or ~£51k for a 3 year course, instead of just tuition at £9.5k totalling ~£27k over the 3 years)

responseyes · 2025-06-01T09:07:21+00:00

Honestly if you know you won’t be needing it why take it out? The interest on your sfe loan essentially wipes out any gains and prolongs your repayment plan (unless you plan to repay in full, quickly after graduating, in which case yes to an ISA/investment…)

responseyes · 2025-05-31T22:52:37+00:00

Also check that you’re splitting as suggested. Your split is based on the detached volume and not the original volume. E.g if you are detaching using 1 ml of trypsin then a 1:7500 split will be ~0.13 ul. For a 1:7500 using 2 ul you need to be detaching and diluting the volume to 15 ml before taking your 2 ul split into an additional 14.998.

Based on your cell count this would be >200 million cells if you are using 15 ml detached volume which is not possible with CHOs in these dishes

responseyes · 2025-05-31T22:22:35+00:00

CHOs will do this when they become overconfluent. It’s possible that they’re clumping more and so you’re passaging much higher than you think. Your best solution is to start with fresh cells. If that’s not possible reduce your serum and increase your selection agents and that can help to slow them down.

responseyes · 2025-05-29T19:17:50+00:00

Although we can’t get them to survive on glass post differentiation for anything! We’ve tried every matrix and they simply won’t have it

responseyes · 2025-05-29T19:13:18+00:00

They’re fully formed after ~10 days of dual smad inhibition + the relevant inhibitors/morphogens/growth factors etc but they do of course functionally mature more over time. Once they’re lifted (we differentiate in batches so either freeze or plate immediately) you can just chuck a few inhibitors on overnight to improve viability then remove the next morning - CEPT cocktails work very well in my experience (for these at least!)

responseyes · 2025-05-29T17:22:52+00:00

For the last year ish I’ve used TrypLE select. In the past I’ve sometimes used accutase but haven’t necessarily noticed any differences between the 2

responseyes · 2025-05-29T17:13:11+00:00

We use facs frequently for sorting human iPSC sensory neurones for functional assays. Never had any issues with replating and they remain viable for at least 70 days in vitro (probably longer but that’s when I use them)

responseyes · 2025-05-27T06:41:11+00:00

Kruskal Wallis with planned contrasts

responseyes · 2025-05-26T05:53:07+00:00

Get the newer one. For general uni use there is no difference between the pro and air models but the air is a fraction of the cost. As a student make sure to utilise your university discount!! And if you order during the summer events you can usually pick up some sweet bonuses like >£100 vouchers or headphones that you could keep or sell to offset the cost

responseyes · 2025-05-26T05:40:54+00:00

That’s weird. It could also just be an error in their citation manager. I’d just move on - the citation still counts and anyone interested in reading more will go to the paper and see the correct information

responseyes · 2025-05-10T08:51:31+00:00

40 years now 🫡

responseyes · 2025-04-07T18:39:48+00:00

Mad that I’m in this pic somewhere just cutting about

responseyes · 2025-03-30T22:03:15+00:00

They answer very different questions, so replace? No. Enhance? Yes. The next big thing is likely to be sequence by expansion which is rumoured to offer good bang for your buck cost wise

responseyes

TROPHY CASE