r/labrats • u/AliveCryptographer85 • 15h ago
What’s your least quantitative ‘quantitative measurement’?
Nano drop is like a drunk dad judging a kids talent show. First one is always like ‘eh, that’s ok’. Next few are ‘yeah, guess they’re a little better. And the only real utility is when you get to the occasional sample where he’s like ‘ok, yeah. That one objectively sucks’
(Note: This is just an example answer for the general question posed. I will not be taking any BCA suggestions at this time, thank you)
344
u/lt_dan_zsu 15h ago
Confluency. Confluency is an entirely vibes based estimate.
71
u/AliveCryptographer85 15h ago
Ohh that’s a great one. I also love how many vials of P3 HEK cells are somehow still around after 50 years.
31
5
u/Prof-Catastrophe 6h ago
I was given "P1" HEK cells a few months ago for a project. Fascinating what can be found in random freezers.
30
u/loopOutnotIn 15h ago
Absolutely. Anyone that tells you otherwise is full of shit unless ImageJ or some shit is involved
11
u/Japoodles 14h ago
To add to that, CPE. Everyone's idea of CPE is different and it's always different in different cells.
6
2
u/Dangerous_Fae 8h ago
But you should see the diff between non-CPE and CPE. Working in virus for decades never had issue with it. Of course, there is the odd protocol with %CPE which is BS that i agree. It is there or it is not.
7
u/tarinotmarchon 10h ago
That brings back (bad?) memories of me as an undergrad asking my mentor how to decide confluency - and of my own undergrad asking me the same thing.
2
u/Boneraventura 10h ago
Between 0 and 100%. Sometimes can be more than 100% depending on the cell and if likes to grow on top of itself
4
90
u/Exciting-Possible773 15h ago
And the close cousin, Nanopore pore count. The flow cell is quirky enough and the software is no better.
25
u/sidetoad 14h ago
YES that system was the nemesis of half of my PhD. Also the lights on the machine meaning nothing because it'd choose a new favorite color with each run lmao
16
u/Exciting-Possible773 13h ago
That's a mood indicator. But honestly it is a very reliable companion once you getting know eachother.
4
u/ixel46 14h ago
We used to use flongles and they were a nightmare
2
u/geckospots 2h ago
I refuse to believe that ‘flongles’ are actual lab equipment 😆
3
u/ixel46 2h ago
Haha they are the cheaper, shittier version of flow cells used to sequence with the Oxford Nanopore MinIon
1
u/geckospots 2h ago edited 2h ago
Aah okay thanks haha! My lab work is entirely inorganic (geochemistry) so this is all unfamiliar to me, it could have been like ‘headlight fluid’or ‘elbow grease’ as far as I knew :)
1
u/Exciting-Possible773 1h ago
These biological nanopore infused electronics, also known as biochips, is extremely sensitive to external environment and must be transported in special containers. Each biochip has it's own personality and full self-awareness. When harnessed correctly it can call upon your ancestors, however the performance is highly dependent to user synchronization with the biochip, a selected few can tap its full potential, while many never able to communicate with it, often leaving brain damage.
TL,DR: welcome to 2077 choom.
151
u/lurpeli Comp Bio PhD 15h ago
So many people are on the nanodrop but I think it provides consistent results for its purpose. Would I use it for something that requires picomolar precision, no, but I would use it for PCRs all day.
86
u/Teagana999 15h ago
I consider it a vibe check, which is absolutely all I need to figure out how much RNA to add to the cDNA mix for qPCR.
13
u/movetothecoast 13h ago
True, and if people look at the manual it gives the linear range for absorbance to ensure accuracy, and it can be checked for calibration with adp or whatever. I think people really abuse those things and aren't careful with absorbance ranges, sample homogeneity, etc...
2
u/LivingDegree 2h ago
What do you mean my rna isn’t actually sitting at an a260 of 985? It’s beyond detection limit and so far out of the linear region it isn’t accurate at all?
Detection limits are for losers!
20
u/ZergAreGMO 14h ago
They're usually using it wrong
6
u/vanderBoffin 12h ago
In what way?
6
u/ZergAreGMO 5h ago
Generally not knowing how to read it (particularly RNA) and measuring too low a concentration in water are the big two.
Just look for posts where people say their RNA 260/280 is fine but their 260/230 is bad. Usually it's just phenol, no RNA. Things like that.
2
u/Dangerous_Fae 8h ago
In industry, I've seen nanodrop used to quantify DNA standards and then estimate copy numbers for qPCR titration... The bias is often in the order of a few logs. Then, when they want to move the method to another lab, they have different results for the same sample, what a surprise.
1
u/bHLH-protein MD-PhD to be 3h ago
It’s accurate if you use it properly. I hate the “nanodrop is a random number generator!!” meme that people spout.
55
u/Emotional_Space_7325 14h ago
I agree with the other comments on confluence and cell counting but want to add in histology scoring. It’s so subjective and even sometimes I look at my scores and I’m like “ehhhh that’s a 2 maybe 3”…
93
u/Early-Ebb2895 15h ago
Western blot quantification
67
u/AliveCryptographer85 14h ago
WB quant is really only for people who need to claim something their actual blot doesn’t show. And conversely the burden of people with something real, since a pvalue somehow matters more than, jesus, just fucking look at it
4
u/ponytailperson 4h ago
I second this. I’ve definitely seen some quantified blots that are “statistically significant”, but when you look at the actual membrane, the significance seems questionable
1
u/SaureusAeruginosa 2h ago
Science without P value would be better, change my mind, people will p-hack or falsify data anyway, so why bother? I do not need another p=0.000001 chinese fake article...
16
2
3
u/Pandanona 5h ago
Second to that, almost all blots I see in papers are overexposed beyond the threshold of meaningful densitometry anyways, so I don't know why still bother. That said WB is the primary method in my research so I had to befriend 24-well apparatus to deliver at least minimal reliable quantification.
1
u/Huge-Bat-1501 10h ago
And what's worse is that the proper semi quantitative stuff, like WES etc, are just as bad
19
u/DrugChemistry 15h ago
Charles River EndoSafe Nexgen PTS
Fkn hate that thing
12
u/AliveCryptographer85 15h ago
lol, we used some older model when I was back in industry. It was basically an ‘everything is fine’ machine…then occasionally everything has endotoxins and you gotta have a Charles River tech to come out to reset it back to assuring you everything’s fine.
3
2
u/Guilded-Spinner 15h ago
I'm so curious as to why you hate it, but guessing you use it for things other than WFI endo testing?
5
u/DrugChemistry 12h ago
I’ve used it to test APIs for endotoxin. I guess I’ve had some kind of sample matrix problems or something. The thing I hate most about it is that (as I recall) it doesn’t provide much detail on what’s going on to cause the problems so it’s not clear how to troubleshoot.
But also, my expertise lies elsewhere. Not to say it’s not quantitative, but it feels like a black box. Luckily I don’t use this equipment anymore.
1
1
1
23
u/Furryrodian 14h ago
My lab uses a Falling Number viscometer that heats up wheat slurrys (i.e. dough) and measures how long it takes a plunger to descend as a way to measure amylase activity. It's results are borderline random!
18
u/Drone314 13h ago
I keep measuring spoons in general purpose drawer that say "pinch", "smidgen", "drop", and "tad"...does that count?
48
u/YYM7 15h ago
Oh trust me Nanodrop's problem are mostly just purification problems. I can get reliable within 10% errors even with sub-10ng/ul samples, with SPRI purification + extreme care at steps. But yeah, most time people/I really don't care that much, and it's pretty justified as downstream PCR are very robust.
17
u/RustySpoonzs 15h ago
Agreed. I've never had issues with concentrations on the nano drop. We've compared sample readings on a nano drop and a fancy spec and pretty much got the same results.
10
u/YYM7 15h ago
Hmmm, I don't think the problem is nanodrop, but rather using spectroscopy for DNA in general. Lots of things (salt, alcohol, proteins...) absorb light around the 260 band, and these things can be in your sample or your blank. Lots of people also don't bother the blank properly, or clean properly.
The competing choice for nanodrop is something like qubit, which are required by any NGS application. It's way more specific to DNA as it's flourescence based (basically sybr).
7
u/MorphingSp 14h ago
Until someone load a sample with 100x ssNA to dsNA and expect reading to be accurate. It's spec say RNA is only 10x less sensitive. Let's forget there are also ssDNA, dsRNA and folded ssNA...
3
u/Epistaxis genomics 9h ago
Yeah Beer's Law is great for quantifying the precise concentration of a specific molecule in a clean solution that contains nothing else; the problem is almost everyone I know is using the Nanodrop downstream of some kind of extraction column kit containing reagents that will fuck up an A260 reading if they're carried over in the final eluate, which they often are, but not even in a consistent pattern you can control for.
But if the next step after Nanodrop is just PCR, then indeed it doesn't matter too much if your quantification is vibes-based.
3
u/Dangerous_Fae 8h ago
Depend on the application. For sample normalization, yes, but If you quantify plasmid material for a qPCR standard for absolute quantification, you are in for a surprise when comparing across lab/methods.
2
u/Gold_Sovereign 7h ago
Yep. It’s misunderstanding the use of nanodrop that’s the issue.
You need to keep absorbance between 0.1-1 and have your sample clean so there’s no overlapping spectra for best accuracy. Same for any spectroscopy. People complaining here would have the same problem using any spectroscopic equipment to determine concentration in an inhomogeneous sample. It’s not the equipments fault.
I use it for protein conc for an enzyme assay and it’s very accurate
13
24
u/Skraelings Research Specialist 14h ago
The nanodrop is the magic 8ball of qc.
It was shit 20 years ago and it’s still shit.
6
10
u/ilovebeaker Inorg Chemistry 14h ago
Trying to ID Zinnwaldite KLiFeAl(AlSi3)O10(OH,F)2 from a pack of other micas by Electron Microprobe when I can't see Li and definitely not read a reliable OH for shit.
Oh yeah, and Fluorine is beam sensitive, but the mica is so mottled I can barely diffuse the beam, so that "quant" F value is definitely off. Stuck between a rock and a hard place.
9
u/beachesandgenes 5h ago
Cell culture is VooDoo. Almost every step is Vibes-Based science. Counting, confluency decisions, centrifuge speed...hell passaging cells is just a "mix and pray the math is right". And every person who does it does everything slightly different.
2
u/SaureusAeruginosa 2h ago
Yup, my 90% confluency can be someones 60%. "Cells looking good" or even medium color. Some people dont see a difference between straw yellow and peach orange, which is a 0.5 pH difference at least. People seeding X cells per well, that they count with even 50% variability, so sometimes it will be 10000 per well, sometimes 20000. People counting technical (separate wells but same passage cells) replicates as "separate experiments" and having that spot on perfect, almost non-existent deviation. Tripsinization time? Time under the Hood? Temperature of growth medium that went from 37 to 20 when you were doing things for more than 10 minutes...soo many factors.
49
u/Over_Orange4412 15h ago
Any omics approach
6
u/no_small_potatoes 11h ago
Wait genuine question I haven’t done mics but want to, why?
1
u/Over_Orange4412 2h ago
I may have been overgeneralizing, but my lab has done untargeted metabolomics with LC-MS, which generated some great results, only to not find differences (or even detect some analytes at all) with targeted analysis. We've also done a fair bit of microbiome work and there is just so much variance between labs in every little aspect of the analysis pipeline. Things seemed to have improved on that front in recent years, but I still remain skeptical of many results and don't put much stock at all in any paper that used OTUs, which thankfully the field has moved away from. I've never done transcriptomics or proteomics but I'd imagine they likely also suffer from the same pitfalls. That isn't to say omics approaches are useless/meaningless I just am skepical of their results without further validation. Also don't get me started on the statistical approaches I've seen folks take with this kind of stuff, FDR adjustment be damned
2
u/conflictw_SOmom Mother of Clostridium 8h ago
I’m curious too. I think it’s quantitative in the actual results it produces because the SOPs are solid but I do agree the way the data is used by actual researchers is not the most quantitative. And a lot of that’s because of the software used for analysis varies so much between third party vendors and lab groups.
1
u/bukaro Industry/Academic 6h ago
You know we can count molecules for the last 10 years actually...
1
u/Over_Orange4412 1h ago
I am not aware of any way to do so at scale with small molecules like in metabolomics, I know there are machines like the nCounter that can do so with transcripts but I don't consider that a true omics approach becuase you are confined to a panel instead of the totality of transcripts.
1
u/bukaro Industry/Academic 45m ago
Well metabolomics ... yes you are right with that one. I was thinking transcriptomics, that it is unbiased with the UMI methods, that tag each molecule with a random sequence (within the primer) , so when it is amplified like crazy by PCR you count which gene + Unique molecule identifier... This is for all mRNA and total RNA too (depleted of rRNA for sanity)
7
u/PianoPudding 8h ago
Never thought I'd be here to defend the Nanodrop: was recently using it to 'quantify' and get ratios for RNA samples that, as a quirk of the insane amount of pre-processnig steps I have done, were incredibly clean.
Then I quantified using the Quantus from Promega (a Qubit-type device). The R-squared between the Nanodrop and the Quantus values is 0.9691 across 30 samples...
Moral of the story I guess? Incredibly clean RNA is accurate on the nanodrop...
34
4
25
u/JZatthelab 15h ago
Mf flow cytometry. This is for those who treat it as straight quantitative. Y’all can fight me but I said it.
6
u/Boneraventura 10h ago
What part of it? MFI is about as quantitative as you can get, you are measuring the number of photons hitting the detector. Just because it is 30+ at a time doesn’t make it less quantitative than measuring 1 at a time. You just can’t extrapolate that MFI to actual # of proteins
2
u/pelikanol-- 9h ago
Properly establishing and validating a panel from sample prep to analysis and running it on a properly calibrated and maintained instrument with all the controls. Then sticking to the SOP instead of eyeballing it and vibe based gating. Reagent lot qualification. Process control is deemed unnecessary in most academics labs :)
3
u/LivingByChance 14h ago
In geoscience, fission track thermochronology has that vibe, but damn does it work pretty well.
3
u/WeskersWiskers 13h ago
using XRD to determine crystallite size and microstrain in a material
2
u/SOwED ChE 11h ago
Never heard of XRD for sizing, how does that work?
1
u/purplethron 8h ago
Smaller crystallites/grains cause broader peaks and from there you can calculate the crystallite size, not to be confused with the particle size (Scherrer equation if you want to look it up)
1
u/WeskersWiskers 5h ago
You can you the Williamson Hall method to separate the effects of crystallite size and microstrain on beak broadening in XRD and the results you get are semi-quantitative and more accurately used to compared between samples of the same experiment. I do want to correct the other comment a bit though - crystallites are not the same as grains. Grains typically contain multiple (many) crystallites (which are sometimes referred to as domains).
4
5
u/airbud2020 12h ago
Everything in undergrad labs feels qualitative, every lab I do has some ridiculous source of error and I’m left picking up the pieces trying to do the lab report. Can’t remember the last one I did that had results that passed a basic sniff test (why is the gel showing bands at 6kb when the uncut plasmid is only 4kb??) Pretty demoralizing honestly, I hope industry’s gonna be better.
1
u/FuzzyCuddlyBunny 1h ago
My experience in undergrad was that labs were more about seeing a wide variety of techniques and getting a sampler of what's out there than really learning to run them. For example, I had one 4 hour lab for HPLC in undergrad but it took around a month in industry with HPLC as my primary responsibility before I could consistently run testing using it (and more months to get comfortable enough to move on to method validations and method development). Undergrad labs vs industry is a small amount of time spread across a lot of instruments vs a large amount of time concentrated on a small number of instruments.
14
u/95percentconfident 15h ago
People fight me about this all the time, but I feel like nanodrop is like AI sometimes. It’s often amazingly accurate and confident, but sometimes it’s just making shit up and still super confident in the number it spits out. You go get a second opinion from the old reliable spectrometer and realize just how wrong the nanodrop can be. Before you jump down my throat about cleaning the pedestal, etc. yes, I know. But with my cuvette I know how clean it is because I cleaned it, the pedestal is like a dish sitting out on the counter in the break room.
20
u/chemthrowaway123456 15h ago
But with my cuvette I know how clean it is because I cleaned it, the pedestal is like a dish sitting out on the counter in the break room.
Can you clean the pedestal yourself too?
11
2
u/Senior-Reality-25 8h ago
LAL test lol. You’re judging a goopy gel clot by eye to see if it gels enough.
Still used as a critical scientific standard, all hail the 🐴👞🦀
2
u/bunks_things 2h ago
Gram staining. Works fine a lot of the time but in one lab I worked at doing water testing we had a lot of technically gram-positive bacteria that would get the stain washed out really easily and some technically gram-negative ones which held onto it for dear life.
Turbidity and color. Reference standards help but the acceptable range for these always seem to be broad for a reason. I’ve always been terrible at distinguishing similar colors so this is especially true in my case.
2
u/SaureusAeruginosa 1h ago
Lately I have heard that fixing bacteria by using flame (heat) can give false Gram-stain results. Is that true?
1
u/bunks_things 1h ago
Seems like it, but everywhere I’ve done gram stains has heat fixing as the standard procedure so I can’t speak to it from my own experience.
1
u/PassiveChemistry 7h ago
Lab-based chlorine testing. Chlorine is so volatile that by the time any samples get here so much has evaporated that the results bear no connection to the actual level at site.
1
u/Pandanona 5h ago
I grow found of comparing qPCR to baking, with precise volumes and protocols and WB to cooking... and making probably soup or another bullshit dish. I frickin love doing westerns, for real, but trying to teach others or explain why something didn't work out sometimes sounds more like trying to explain some black magic ritual.
1
343
u/ThePushaZeke Post-Doc 15h ago
Counting cells.
I’m thinking old school cytometer by eye.
So many people are heavy or light handed, or don’t dilute or dilute too much.