Would you believe they put a man on the moon?

A brief departure from the story that still needs telling...

Today, July 20th, 2008, marks the 39th anniversary of the Apollo 11 moon landing (July 20, 1969). The mission accomplished what John F. Kennedy dared the country to achieve over 9 years earlier in 1960. The mission was historic on many levels (first human landing on an extraterrestrial body being perhaps the most significant of them), and yet it saddens me that many people doubt it ever happened and believe that our presence in space is now just fulfilling some antiquated and somewhat misguided dream.

What about all the problems here on Earth? some will argue. Shouldn't we be more concerned with ending poverty, curing infectious diseases, stopping wars, etc than spending tax dollars to send up yet another space shuttle, yet another satellite, yet another telescope, yet another human being on the greatest adventure our species has ever undertaken.

To leave one's home is a universally recognized sign of maturity and independence. Species that travel around in troops (chimpanzees for example) routinely send out either male or female members once they "come of age" to find another troop to join, thus keeping the gene pool well mixed and reducing friction at home. For mankind to leave Earth's land, sea, and air is a feat achievable by no other extant species and one accomplished by no extinct species either. Nature abhors a vacuum and thus no known living thing can exist in the harshest of all environments. Even Thermus aquaticus, a hardy bacterium whose ability to thrive and survive in water at temperatures between 160 and 175 degrees Fahrenheit allowed for the development of polymerase chain reaction technology (discussed in a an earlier post) that has revolutionized modern biology could not survive in a vacuum. Even the phantasmagoric creatures that inhabit the darkest depths (miles down) of the ocean where pressures can be hundreds of times what we feel at the Earth's surface could not survive in a vacuum.

But lauding the merits of the manned space program is a topic for another day. For now, the 39th anniversary of the moon landing should be a reminder that even the seemingly impossible can be achieved in a short amount of time if there are enough dedicated minds, hearts, hands, and dollars to the cause. This week, ex-Vice President Al Gore delivered a challenge reminiscent of JFK's proclamation to the nation nearly 50 years ago.

Gore delivered his address at a time of impending economic recession, two wars without end, a presidential election year, a disastrous housing crisis, long-delayed recognition of the crumbling infrastructure throughout the nation, genocide in Darfur, nuclear fears in Iran and North Korea, possible war between Thailand and Cambodia, increasingly armed and friendly China and Russia, and the weakest dollar in decades. In spite of all these ills, Al Gore wants us to save...the environment? Now why on earth should be worried about the Earth's health and condition when our own health and standard of living is being threatened by so many forces? Why? Because of a simple idea few seem to recognize with any immediacy or profundity: without a healthy planet there can be no healthy life or adequate standard of living for any of us, no matter how rich or entitled we may be.

If people in America think gas prices are bad now (~$4 a gallon), imagine how they'll feel when those oil reserves start drying up and everyone, including people in China, India, and Africa once disregarded as potential consumers in older calculations, is still clamoring for more and more oil. But if we can free ourselves from the chains of petroleum we can not only avoid that kind of energy crisis, but more importantly remove the yoke of OPEC and its member dictatorships from around our neck and breathe the air of freedom certain individuals in our administration are trying so hard to instill in many of those nations.

So how does the environment factor into all this? It's no coincidence that "environmentally friendly" practices (from waste disposal to energy generation to transportation, etc) use fewer resources of all types, including energy. The best way to eventually do away with our dependence on fossil fuels is to reduce that dependence first. Reduce, reuse, and recycle. Those simple three R's that everyone's heard but few truly heed. For far too many people:

#1 unthinkable--> I want more stuff, not less.
#2 distasteful--> I want new stuff, not old stuff.
#3 inconvenient--> I can't spare the time or effort to sort my trash, and there are no recycling bins near me.

Al Gore is leading a movement to change this way of thinking, because without a new trend of environmentally conscious behavior the planet and all its inhabitants will turn into the dystopia pictured in WALL-E.

Enough ranting. The moral of my story: let's do for the Earth what we did for man in space. If we succeed in this venture, future generations won't have to doubt that our mission was ever accomplished. They will be living proof it did.

Saturday, 14 Jun

I woke up earlier this morning than usual for a very special reason....the family was going to arrive soon! No sooner do I get out of the shower and am ready to wash my hair I get a phone call from Sachi saying they're about a half hour away from the city and would call again to clarify directions as they needed them. I quickly dried my hair, got dressed in my brand new UChicago shirt and after informing Bonnie and Jeffrey to expect my fam, I skipped down the steps to wait outside. Fortunately, it was a glorious day. Now the definition of "glorious' weather obviously varies from person to person, but I feel few could have argued with my definition today. Fluffy, sculpted clouds drifted lazily across and otherwise perfectly empty azure sky; a gentle breeze stirred the leaves whose whispers were frequently interrupted by chirps from robins to cardinals and other species I couldn't recognize. The temperature was perfect in the lower 70s F. In short, it was truly a glorious day and quite a pleasant change from the 90+ temps they were leaving back in Cary, NC.

I scan the streets in all cardinal directions for our gray Odyssey and after a few minutes spot one a block over heading too far north. I'm sure it's them and laugh because they clearly missed their turn but will realize it soon. Sure enough, a few seconds later I get a call from Sachi informing me they went a little far and am heading back down to me. Finally, the van cruises down Greenwood Ave and grinning from ear to ear I guide them to a good parking spot. Reunited at last!! I'm engulfed in a succession of bear hugs from everyone and then it's time to unload and head upstairs. So much stuff! It feels like I'm moving in all over again. B&J give my folks a warm welcome and while they all chat, I drag the goodies into my room to start unpacking. Chuckrie, shrikhand, kofta, paneer, nuts of all kinds, my Indiana Jones hat!, the treats just keep coming...

After driving so long, I knew they wouldn't mind some exercise so I take them on a jaunt around campus and my lab (the one I was officially leaving but still had a key, and permission, to give a tour). I forgot this was also graduation weekend and so the quad was filled with students in black gowns, parents in their Sunday best, and lots of food and drinks under tents. Thinking that usually big crowds = full restaurants I hurry us over to Noodles, Etc to beat the people I'm sure would be hot on our heels. Fortunately, the place was only half full and we get a nice spot by the window. Sure enough, no sooner do we get our orders then the place starts filling up with all the grads and their families. Whew! That was a close call. After eating, I thought walking down to the lake might be nice so we take a nice stroll along 57th street until we cross over to the greenbelt.

We relax on the rocks for a bit and then head back home along 53rd this time so they could see one of the other major commercial streets in Hyde Park. It's tea (and nap for Mom) time at home so we brew up some refreshments and sit around chatting for a bit. At the same time, my neighbor (and professor) Marcus Peter was having a celebration downstairs to mark his son's graduation, daughter's birthday, and his and his wife Andy's new US citizenship. After everyone was rested, we head downstairs for a little while so that Marcus could meet my folks. I see a couple other professors (including one of my favorites, Kay MacLeod) there and introduce them to my folks. It's nice to see them in a completely non-academic context.

After hanging out at the party for a bit it's time to pack my things and head into town before going to our hotel (six people in a two-bedroom, 3 person place for a week would have been a bit much, and it will give B&J some time alone that they haven't really had since I moved in). Sachi found a placed called The Italian Village in downtown Chicago that was a bigger deal than I think any of us realized until we got there. The layout was spectacular: lots of little booths and alcoves that both created a sense of space and made the restaurant feel more intimate...rather paradoxical but it worked. We are promptly led to a little alcove and enjoy a fine meal without the crowd and noise one is used to in restaurants. This place takes the idea that ambience and decor can make or break a dining experience seriously and to wonderful effect.

Dinner over, we drive out to our hotel which is about 30-40 minutes from the city. We're pretty beat from a long day so tuck in early and call it a night.

End of Term and the Family (most of it) Visits

Finally, spring quarter officially ended for me on June 13th. Although classes were over the week before, as I mentioned I spent exam week in lab so technically my quarter didn't end until I finished that last real-time. Grades-wise I fared pretty well; two passes, one A, one B, and one (the rotation) I don't have the results for yet. B's are common, A's are rare, so we'll see.

But let's forget about school work for now...and until September (except for research which I'll be discussing periodically). The following posts will chronicle the week from June 13th to the 21st that I spent with most of my family, i.e. Sachi and my parents, in and around Chicagoland [Saket, that poor guy, was starting his first full week at Apple, Inc out in the Valley at a sweet internship with their iPod hardware division...yes, let's all feel very sorry for him :)].

Success at last!

If you recall, on my last post I had said that none of my experiments had really worked. Well, as of Friday the 13th, all of that changed...

In my final week in Dr. Dolan's lab, which also coincided with finals week (which wasn't a big deal since I only had one take home final to work on), I decided that it would be worthwhile to repeat my knockdown experiment one last time, except with a few changes: 1) shorter time point of 6 hours vs. 24 hours, 2) added the siRNA to the nucleofection 96-well plate before adding the cells+NFS mixture, 3) allowed the cells to rest for 10 minutes at room temperature in the hood before adding pre-warmed media after the nucleofection, and 4) pooled four wells per sample instead of two.

On top of changes to the protocol, my technique was solid on the RNA isolation, RT-PCR, and real-time PCR. My RNA yield was the best it'd been all quarter, and my real-time results were not only beautiful but more importantly were believable because the standard curves (for the standards and samples) had a slope of about -3.4 which is much closer to the ideal -3.3 that's expected for totally pure cDNA.

So what was the final result? At the highest concentrations tested (40 and 60 pmol), I saw an almost 80% decrease in CYP1B1 RNA for both cell lines after 6 hours of transfection. These results are very exciting since it shows that transfection is possible with these LCLs (otherwise considered a very difficult system in which to transfect siRNA) and that the vector system we've chosen (96-well, the particular program and NFS) are workable, if not necessarily optimal, choices.

The next step is to carefully note any and all the changes to the protocol I made (some of which were really just reverting back to what Amaxa, the supplier of the transfection stuff, recommended) and then explain those to others in the lab. Woohoo!

Real-time PCR

So I've taken you from cells to RNA to DNA, now it's time to find out whether my siRNA knockdown experiment actually worked. What real-time PCR does is implied in the name; it provides information about the quantity of a particular gene (or genes) at the time the assay is performed. The PCR part comes from the fact that while during the reverse-transcription step we use random primers to amplify all the RNA in our sample, this time we want to only amplify particular genes so we use special primers for them. What makes these primers special? Let's say they give the reaction a special glow.

A company, Applied Biosystems, has developed a system (or perfected it), in which a special probe with a colored dye and a quencher (a compound that masks the dye) is affixed to one end of the primer (a small stretch of DNA that will anneal to one end of a gene of interest) such that when the DNA polymerase enzyme that adds DNA nucleotides together runs into it, the probe is cleaved and the dye and its quencher are separated such that when the sample is excited, light is emitted which is read by a detector. This cleavage, excitation, and light emission occurs during each cycle, so the more of the target DNA there is, the more light will be emitted and thus read by the machine. Make sense? Multiple dyes for multiple genes can be added to each sample. In our case, we want to know the levels for our gene of interest, CYP1B1, and a housekeeping "standard" gene (HKG), huB2M, that lets us know the quality of cDNA we're testing and to control for any extra high overall gene expression in our cells (which is a frequent issue with tumor cells, though our LCLs are from normal, healthy individuals).

Once the samples are read, fold-change is assessed by dividing the amount of CYP1B1 by the amount of the HKG, and then dividing our concentrations by the 0 pmol (control) sample. If things work as they should, we expect to see a dose-dependent decrease in CYP1B1 in cells receiving the siRNA such that the higher the concentration of siRNA added, the lower the resulting gene expression.

My experiments, unfortunately, did not work. I got crazy fold-change numbers such that for one cell line I first saw a knockdown of about 40% and then two weeks later saw a 1×10^7 (that's 10 million fold) increase in expression! Crazy. Naturally, I repeated the experiment and will get my results tomorrow. Later!

RT-PCR

"Reverse transcription polymerase chain reaction" is a five-word mouthful for something more easily said in four simple words: "turning RNA into DNA." The polymerase chain reaction (PCR) single-tubedly revolutionized molecular biology and changed the pace of research in the field forever. It was such an important development that like many other important scientific techniques (including electron microscopy and centrifugation (here called "disperse systems)), it won the Nobel Prize in 1993 and rightfully so. In short, what PCR does is turn a very small amount of DNA into a lot of DNA, still microscopic of course but enough that scientists can actually work with and manipulate it. Here's a video of the process.

The "reverse transcription" means a lot of what it implies. Just as I said how transcription was turning DNA into RNA, the reverse of that process turns RNA into DNA. Viruses are unique in terms of "living" things (viruses are technically not considered living because they need a host in which to reproduce) because some of them use RNA (single stranded or double stranded) as their genetic blueprint instead of DNA (like we and every other organism in every kingdom of life do). HIV is one such example of an RNA-virus. As suggested, these viruses have the natural ability to turn RNA into DNA, a process they need to do in order to integrate their genetic material into that of their hosts'. To do our research, we borrow the special enzyme they use called "reverse transcriptase." It's actually quite remarkable how many uses scientists have for viruses and their products.

So for RT-PCR, instead of the normal DNA polymerase (an enzyme that joins DNA nucleotides together), we use reverse transcriptase. And since we want to convert all of our RNA into DNA (all of the genes), we use random primers (short sequences of nucleotides that specific a part of the genome to amplify) instead of specific ones (those come into play later when we hone into one gene).

Unlike the laborious process of RNA isolation, RT-PCR is quite simple and is just me making up a mix, aliquoting it into tubes with my RNA, then throwing all those tubes into a machine that does the heating and cooling (cycling) for me according to a program a lab mate set up. All I have to do is come back in a couple hours and boom, collect my DNA. Kinda like magic. Then again, a lot of science seems like magic....

Next up: Real-time PCR!

RNA Isolation

So I left off talking about we artificially reduce protein levels (be reducing levels of the template) and what we hope to see from that. Now, briefly, I'll describe how we can tell those protein levels have gone down to the extent that we want them to.

First step, isolating the total RNA from the cells. Think of this step as a lot like panning for gold. You're standing in the middle of the river, with your pan in hand, and scoop up a good mound of the river bed. In it are pebbles, sand, tiny microorganisms, some plant matter, maybe some pieces of pollution, and of course some tiny nuggets of gold. The quality of the gold doesn't matter right now, you just want everything with Au atoms in it. So how do you separate the yellow stuff from everything else? First you shake the pan rather vigorously, then as more and more of the junk falls out you sift more slowly and carefully, occasionally washing the pan with water. Finally, after shaking and washing, you see clinging to your netting tiny little nuggets of gold that you then store into well sealed containers for later analysis.

And now back to RNA. Much like the example, RNA isolation is a process that ranges from the rough and tumble to soft and gentle. Cells from the nucleofection plate are pipetted up, put into tiny centrifuge tubes, spun down at about 100g until they form a pellet, and are dropped into liquid nitrogen where they are flash frozen. (By the way, liquid N2 remains hands down the coolest reagent in science. Though I've handled it many times now, it never gets old).

After the cells are frozen (in which all the liquid around them gets frozen or evaporated off too), they are resuspended in a solution that breaks up the cells to release all their contents. The tubes are spun again so all the heavy stuff (proteins, etc) sink to the bottom while the light stuff (DNA, RNA), stay at the top. The tubes we use are two-chambered in that the bottom chamber collects eluent that flows down from the top chamber. Near the middle of the top chamber is a special filter to which nucleic acids (DNA and RNA) can cling but nothing else can. This way it is easy to discard everything but the nucleic acids throughout the process.

Once protein and nucleic acids are separated, it's time to get rid of DNA since we're not interested in it. We use a tube that has a specific filter for DNA that traps it while letting RNA through. Keep in mind none of these steps are perfect, but these macromolecules are different enough such that these separation methods really work quite well. To get just RNA, it's a matter of washing with various solvents and spinning the tubes many times in a centrifuge. At the end, we get about 2.5 or more micrograms of RNA (that 2.5 millionths of a gram). Very, very small amounts of material here. Once we've got our RNA suspended in water, we throw into the freezer at -80 C until we're ready for the next step: reverse-transcription polymerase chain reaction, also known as RT-PCR.