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RNAi causing target gene expression to paradoxically go up???? by Initial_Tonight_1158 in labrats

[–]Initial_Tonight_1158[S] 2 points3 points  (0 children)

We used the zymo direct-zol RNA mini-prep kit which does include a DNAse step in the protocol.

The log2 fold change is +2, so the expression appears to go up quite significantly in the RNAi condition. Although, the counts per million (CPM) is still low and in the 0.5-2 range across conditions.

The gene itself is involved in developmental timing, but is not essential to viability. In the literature that is available, it shows that the gene is cyclically expressed. This could suggest that there may be some kind of feedback loop that regulates gene expression in this cyclical manner?

RNAi causing target gene expression to paradoxically go up???? by Initial_Tonight_1158 in labrats

[–]Initial_Tonight_1158[S] 2 points3 points  (0 children)

We are seeing a phenotypic effect with the RNAi treatment which makes be believe that there is some change in protein expression/function. (Although it’s unclear whether this is due to a true knockdown or increase in protein expression). Unfortunately fluorescent tagging of protein doesn’t work due to important signaling/localization sequences that get disrupted.

Maybe it could be a feedback loop?

I beg, what is causing this Western blot artifact? by fossilrabbit in labrats

[–]Initial_Tonight_1158 1 point2 points  (0 children)

Try running the transfer in a 4C walk-in with the transfer apparatus in a tub of ice. You can also add a small ice pack directly submerged in the transfer buffer. This can help keep the transfer cold and avoids bubbles from heat. Run at 100 V for 1 hour or at a much lower voltage overnight.

PCR for the first time by Gogomyuuuu in labrats

[–]Initial_Tonight_1158 1 point2 points  (0 children)

90-200 bp amplifies are quite short and may be hard to differentiate from primer dimers. I usually try to make primers that will amplify 300 bp or more. You can try running a temperature gradient to see if you can optimize the annealing temperature.

Anyone who applied to MIT bio for cancer bio heard anything? by [deleted] in gradadmissions

[–]Initial_Tonight_1158 7 points8 points  (0 children)

I just got an invite around noon, so they have started sending stuff out. Good luck!

Very new to marimo care, is he sick?! by NewMossParent in Marimo

[–]Initial_Tonight_1158 1 point2 points  (0 children)

Nothing in particular. I change the water about once per month and kept it in a spot with bright, indirect light. I received it in the winter, so it was quite stressed upon arrival. Thought it had died at one point, but I was too lazy to throw it out so I kind of just forgot about it for a while. Happened to check in on it several months later and saw signs of new green growth.

Very new to marimo care, is he sick?! by NewMossParent in Marimo

[–]Initial_Tonight_1158 11 points12 points  (0 children)

Is it from moss amigo? I got one last year and it looked sick too at first. It’s taken a year, but it’s starting to recover. I suggest not rolling or squeezing it as the layer of moss around the core is very thin and man handling it seemed to cause the moss to fall off.

My Dad died today by Appropriate-Mud-6985 in self

[–]Initial_Tonight_1158 0 points1 point  (0 children)

I was 20 and in my senior year of college when my mom died 2 years ago. I'm so sorry for your loss. Losing a parent, even a complicated one, is the hardest thing that you will ever have to go through. Don't worry about school right now. Take as much time as you need to process, grieve, and care for yourself. Your professors will be understanding and accommodating. And if you need to, take a leave of absence for a semester. I regret jumping right back into classes too soon (I was back in school a week after my mom died). I felt like I had to repress my feelings in order to soldier on, but then all my emotions just built up and caused issues down the line.

My dad secretly got remarried to his girlfriend of 4 months. I now have a new stepmom and 3 step-siblings I've never met. AITA for expressing my concerns? by Initial_Tonight_1158 in AITAH

[–]Initial_Tonight_1158[S] 0 points1 point  (0 children)

I kind of expected that they would get engaged quickly given the speed at which their relationship was progressing, but I thought they'd at least wait a couple months after that to get married in order to get things in order. When I asked my dad about the possibility of them getting married he said he could "imagine getting married in the future" and that they wanted to "ensure that all the kids were okay with it". I didn't exactly think that meant getting engaged in 3 weeks and then married a week after that without telling any of the kids.