I have worked with human placentas for gene expression analysis. You need to process the placenta within the first 2-3 hours of birth (speed is important). We used dissecting scissors and cut out two full thickness sections (going from the maternal side all the way down to the fetal side) with a diameter of 0.5 - 0.8 cm were cut out. These were then medially dissected between the maternal and fetal side. Then you need to wash the tissue with PBS, blot it dry and transfer it to a clean labelled pot. 

The tissues destined for RNA processing were placed in tubes with RNAlater solution that stabilises and protects the cellular RNA. These were finely minced with scissors to increase surface area of the tissue exposed to the solution. Then the tissue was transferred to the research facility, removed from RNAlater and into another tube and frozen in -80C or liquid nitrogen until the time for extraction of RNA (using trizol or a kit) or isolating MSCs.

Here is a published protocol for the isolation of amniotic  MSCs which might help you in isolating it from the placenta.

Thank you Sophia Malandraki-Miller

    I want to know what is the best method to transport human placenta to my lab and stored to get good results in RT-PCR ( For example, for IHC assays , placenta wash with phosphate buffered saline then injected with formalin through the umbilical vein, preserved in formalin, and then transferred to the lab but in RT-PCR , not helpful put them in formaldehyde and stored until assays perform ) isn't it ?! .

For RT-PCR , i will follow these steps .

1- RNA isolation by RNeasy Mini kit including DNase I digestion following the manufacturer's protocol (QIAGEN, # 74104, Germany).

2- for RNA reverse transcribtion use Quantiscript reverse transcriptase (QuantiTect Reverse Transcription Kit, QIAGEN, # 205310, Germany) then quantitative real-time PCR analysis performe with a (Rotor-Gene Q 2plex, Qiagen, Germany) with SYBR Green master mix (Roche Diagnostics).

3- The gene expression normalization to human β-actin. The following thermal cycling conditions use for the four genes ( my genes ) : initial denaturation at 94 °C for 3 min, 35 cycles of amplification (DNA denaturation at 94 °C for 40 s, annealing at 56-58 °C for 1 min, extension at 72 °C for 1 min) and final extension at 72 °C for 10 min. The real time thermocycler (Rotor-Gene Q 2plex, Qiagen, Germany) use for this purpose. The mRNA concentrations of all detecttion genes is normalization and represente as a ratio against β-actin. The software (Rotor Gene Q, version 2.0.2, Build 4) used for data analysis.

What do you think , is it good protocol?!

Regards

 There are number of kits available for simultaneous isolation of DNA, RNA and Protein from cell lines or fresh tissue.  Here is one protocol from Nature : http://www.nature.com/nprot/journal/v8/n11/full/nprot.2013.141.html

There are private companies like Sigma, QiaGen and few others, who also sell these reagent kits.

I can meet you next week to give you few other options?

Regards 

Biopsy through minor surgical procedure or suction through syringe

http://www.primerdesign.co.uk/assets/files/beginners_guide_to_real_time_pcr.pdf

I think it will helps you

Hi,

You should adopt a nine-site sampling protocol for both foetal and maternal side of the placenta. In nine-site sampling you divide the placenta into 9 (or more!) areas and then extract a sample from middle of each site. Sample into cryo-tubes labeled with 1-9 so you can locate your sample site later on and use for example tube 5 for the central piece for comparable analyses between the placentae. There are punch tools for tissue sampling, that provide you with a comparable amount of tissue across samples. Samples should be obtained within 2 h window post partum and stored in sufficient RNA-later and subsequently frozen until RNA extraction.

For placenta guidelines check out:

Burton GJ, Sebire NJ, Myatt L, Tannetta D, Wang YL, Sadovsky Y, Staff AC,

Redman CW. Optimising sample collection for placental research. Placenta. 2014

Jan;35(1):9-22. doi: 10.1016/j.placenta.2013.11.005. Epub 2013 Nov 19. Erratum

in: Placenta. 2014 Apr;35(4):289. PubMed PMID: 24290528.

regards,

Inkeri Lokki

University of Helsinki

I have worked with human placentas for gene expression analysis. You need to process the placenta within the first 2-3 hours of birth (speed is important). We used dissecting scissors and cut out two full thickness sections (going from the maternal side all the way down to the fetal side) with a diameter of 0.5 - 0.8 cm were cut out. These were then medially dissected between the maternal and fetal side. Then you need to wash the tissue with PBS, blot it dry and transfer it to a clean labelled pot. 

The tissues destined for RNA processing were placed in tubes with RNAlater solution that stabilises and protects the cellular RNA. These were finely minced with scissors to increase surface area of the tissue exposed to the solution. Then the tissue was transferred to the research facility, removed from RNAlater and into another tube and frozen in -80C or liquid nitrogen until the time for extraction of RNA (using trizol or a kit) or isolating MSCs.

Here is a published protocol for the isolation of amniotic  MSCs which might help you in isolating it from the placenta.

Hi Sqaleh Alkarim

I think that you have two options to transport  nd collect your samples

Snap frozen I liquid nitrogen or collect the samples in RNA-later.

The RNA-isolations kit you are suggested is good, I have very good experience of them.

However my experience with Syber green RT-PCR are not that good. I suggest that you use for examples Taqman assays with a minor groove binding probe (MGB). I will also suggest that you use more than one house keeping gene Gapdh and GUSB. Try to find a house keeping gene with a similarly CT-value as the genes you will study.

We are using Invitrogens one step kit for RT-PCR, SuperScript® III Platinum®One-Step qRT-PCR Kit.

Good Luck

Hi,

Ive worked with human placenta for the last 20 years.  AS others have said to get high quality RNA from the placenta ( which has a lot of endogenous RNases) I recommend processing between 30mins to one hour after delivery.  I am providing the link to our biobank SOP. We freeze our samples in liq N2, RNA later is good too, just make sure you wash tissue well and use enough RNA later and small pieces of tissue.  To get high quality RNA from placenta we crush the tissue with a mortar and pestle on dry ice, (tissue must be kept frozen until in RNA extraction buffer).  I recommend using TRizol, for the original isolation it copes with the blood much better than the Qiagen kits.  For RT PCR we do Dnase 1 in solution and then clean up on the RNeasy Midi Kit.   We routinely use SYBR green PCR, house keeping genes for placenta are CYC1, TOP1, TBP, YWHAZ, HPRT, SDHA.  http://biobank.lunenfeld.ca/?page=Custom Services  for the SOP for placental collection.

Good luck Caroline